Chip-based isothermal amplification devices and methods

ABSTRACT

Disclosed are methods and compositions for isothermal amplification of nucleic acids in a microfabricated substrate. Methods and compositions for the analysis of isothermally amplified nucleic acids in a microfabricated substrate are disclosed as well. The microfabricated substrates and isothermal amplification and detection methods provided are envisioned for use in various diagnostic methods, particularly those connected with diseases characterized by altered gene sequences or gene expression.

[0001] The present application claims the priority of co-pending U.S.Provisional Patent Application Serial No. 60/031,590, filed Nov. 20,1996, the entire disclosure of which is incorporated herein by referencewithout disclaimer.

[0002] The government owns rights in the present invention pursuant togrant number R01-HG01044 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the field of molecularbiology, and relates to methods for amplifying nucleic acid targetsequences in microfabricated devices. It particularly relates toisothermal methods for amplifying nucleic acid targets inmicrofabricated devices. The present invention also relates to methodsof detecting and analyzing nucleic acids in microfabricated devices.

[0005] 2. Description of Related Art

[0006] In vitro nucleic acid amplification techniques have providedpowerful tools for detection and analysis of small amounts of nucleicacids. The extreme sensitivity of such methods has lead to theirdevelopment in the fields of diagnosis of infectious and geneticdiseases, isolation of genes for analysis, and detection of specificnucleic acids as in forensic medicine.

[0007] Nucleic acid amplification techniques may be grouped according tothe temperature requirements of the procedure. Certain nucleic acidamplification methods, such as the polymerase chain reaction (PCR™—Saikiet al., 1985), ligase chain reaction (LCR—Wu et al., 1989; Barringer etal., 1990; Barony, 1991), transcription-based amplification (Kwoh etal., 1989) and restriction amplification (U.S. Pat. No. 5,102,784),require temperature cycling of the reaction between high denaturingtemperatures and somewhat lower polymerization temperatures. Incontrast, methods such as self-sustained sequence replication (3SR;Guatelli et al., 1990), the Qβ replicase system (Lizardi et al., 1988),and Strand Displacement Amplification (SDA—Walker et al., 1992a, 1992b;U.S. Pat. No. 5,455,166) are isothermal reactions that are conducted ata constant temperature, which is typically much lower than the reactiontemperatures of temperature cycling amplification methods.

[0008] The SDA reaction initially developed was conducted at a constanttemperature between about 37° C. and 42° C. (U.S. Pat. No. 5,455,166).This was because the exo klenow DNA polymerase and the restrictionendonuclease (e.g., HindII) are mesophilic enzymes that are thermolabile(temperature sensitive) at temperatures above this range. The enzymesthat drive the amplification are therefore inactivated as the reactiontemperature is increased.

[0009] Methods for isothermal Strand Displacement Amplification, whichmay be performed in a higher temperature range than conventional SDA(about 50° C. to 70° C., “thermophilic SDA”), were later developed.Thermophilic SDA is described in European Patent Application No. 0 684315 and employs thermophilic restriction endonucleases that nick thehemimodified restriction endonuclease recognition/cleavage site at hightemperature and thermophilic polymerases that extend from the nick anddisplace the downstream strand in the same temperature range.

[0010] Photolithographic micromachining of silicon has been used toconstruct high-throughput integrated fluidic systems for a variety ofchemical analyses. This technology is of particular interest for thedevelopment of devices for analysis of nucleic acids, as in theirconventional formats such analyses are typically labor- andmaterial-intensive. Ideally, all of the processing steps of theamplification reaction would be conducted on the microfabricated deviceto produce a completely integrated nucleic acid analysis system forliquid transfer, mixing, reaction and detection that requires minimaloperator intervention.

[0011] Silicon and glass devices are economically attractive because theassociated micromachining methods are, essentially, photographicreproduction techniques. Silicon structures are processed using batchfabrication and lithographic techniques. These processes resemble thoseof printing where many features may be printed at the same time. Theseprocesses permit the simultaneous fabrication of thousands of parts inparallel, thus reducing system costs enormously. Today, siliconfabrication techniques are available to simultaneously fabricatemicrometer and submicrometer structures on large-area wafers (100 cm²),yielding millions of devices per wafer and may be used to process eithersilicon or glass substrates.

[0012] These characteristics have led to the proposal of silicon andglass as a candidate technology for the construction of high-throughputDNA analysis devices (Woolley and Mathies, 1994; Northrup et al., 1993;Effenhauser et al., 1994). As mechanical materials, both silicon andglass have well-known fabrication characteristics (Petersen, 1982).Microfabricated devices for biochemical and fluidic manipulation areundergoing development in many laboratories around the world (Ramsey etal., 1995; McIntyre 1996). Over the past 10 years, a number ofmicrofluidic devices have been developed that allow the construction ofminiaturized “chemical reactors.” Individual components of the systemsuch as pumps (Esashi et al, 1989; Zengerle et al., 1992; Matsumoto andColgate, 1990; Folta et al., 1992); valves (Esashi et al., 1989,Ohnstein et al., 1990; Smits, 1990); fluid channels (Pfahler et al.,1990); chromatographic and liquid electrophoresis separation systems(Terry et al., 1979; Harrison et al., 1992b-g; Manz et al., 1991; Manzet al., 1992) are available. Although an objective of several researchgroups, complete silicon-fabricated nucleic acids analysis systems arestill at the earliest stages of development.

[0013] Other components that have been microfabricated which areapplicable to nucleic acid analysis include elements for gelelectrophoresis (Zeineh and Zeineh, 1990; Heller and Tullis, 1992;Effenhauser et al., 1994; Woolley and Mathies, 1994, 1995; Webster eta., 1996); capillary electrophoresis (Manz et al. 1992, 1995;Effenhauser et al., 1993; Fan and Harrison, 1994; Jacobsen et al.,1994a; 1994b; Jacobson and Ramsey, 1995; Ocvirk et al., 1995; von Heerenet al., 1996); synthetic oligonucleotide arrays (Fodor et al., 1993;Schena et al., 1995; Hacia et al., 1996); continuous flow pumps (Lintel,1988; Esashi et al., 1989; Matsumoto and Colgate, 1990; Nakagawa et al.,1990; Pfahler et al, 1990; Smits, 1990; Wilding et al., 1994; Olsson etal., 1995); discrete drop pumps (Burns et al, 1996); enzymatic reactionchambers (Northrup et al., 1994; Wilding et al., 1994b; Cheng et al.,1996); optical/radiation detectors (Belau et al., 1983; Wouters and vanSprakelaar, 1993; Webster et al., 1996); and multicomponent systems(Harrison et al., 1992, 1995; Northrup et al 1994; Jacobsob and Ramsey1996).

[0014] To date, a number of devices have been micromachined, includingpumps and valves (Gravensen et al., 1993; Manz et al., 1994; Colgate andMatsumoto, 1990, Sammorco et al, 1996); reaction chambers (Woolley andMathies, 1994; Wilding et al., 1994); and separation and detectionsystems (Weber and May, 1989, Northrup et al., 1993, Harrison et al.,1993; Manz et al., 1992; Jacobson et al., 1994; Schoonevald et al.,1991; Van den Berg and Bergveld, 1995; Woolley et al., 1995). Some ofthese have been recently integrated together to build pharmaceuticaldrug closing systems (Lammerink et al., 1993; Miyake et al., 1993) andother microchemical systems (Nakagawa et al., 1990; Washizu, 1992; Vanden Berg and Bergveld, 1995). One device is an integrated glass systemcombining DNA restriction enzyme digestion and capillary electrophoresis(Jacobson and Ramsey 1996). An alternative format using high-densityarrays of synthesized oligodeoxynucleotides has been demonstrated as aDNA sequence detector (Fodor et al., 1993; Hacia et al., 1996).

[0015] Nucleic acid targets have been successfully amplified by the PCR™on such microfabricated devices, often referred to as “chips” (U.S. Pat.No. 5,498,392; Woolley et al., 1996; Shoffner et al., 1995; Cheng etal., 1996; Wilding et al., 1994; U.S. Pat. No. 5,589,136; U.S. Pat. No.5, 639,423; U.S. Pat. No. 5,587,128, U.S. Pat. No. 5,451,500) and LCR(Cheng et al., 1996; U.S. Pat. No. 5,589,136). Evaporation due torepeated exposure to high temperatures during thermocycling is aproblem. Evaporation during PCR™ has been controlled by immersing thechannel in oil such that the open ends are covered, but this makesrecovery of the amplified sample difficult.

SUMMARY OF THE INVENTION

[0016] The present invention overcomes the foregoing evaporation andrecovery drawbacks, and other deficiencies inherent in the prior art, byproviding compositions and methods for use in the isothermalamplification of nucleic acids in microfabricated devices. In contrastto the difficulties previously perceived to exist and the prejudices inthe art, the inventors found isothermal amplification of nucleic acidsusing microfabricated devices or “chips” to be surprisingly effective.In fact, the chip-based isothermal amplification of the presentinvention was discovered to be efficient at previously untested lowtemperatures, despite potentially negative effects of surface chemistryand other proposed problems, such as stagnant temperature gradients,reduced diffusion and mixing, and inhibition of enzyme activity.

[0017] The invention thus generally provides an apparatus, system,device or chip, or a plurality thereof, with isothermally regulatedreaction chambers, methods of constructing single-chip and multiple-chipanalytical systems, and methods for using such devices, chips andsystems in the isothermal amplification of nucleic acids. The inventionalso provides for the analysis of the amplification products using,e.g., sequencing, gel separation, and/or detection of the amplificationproducts in microfabricated devices. Further methods of the inventiontherefore include laboratory methods connected with nucleic acidanalysis and clinical methods connected with the diagnosis and prognosisof disease states.

[0018] First provided by the invention are devices, chips, wafers or ananalytical apparatus or system(s), generally of a microfabricated ormicromachined type, for use in the isothermal amplification of selectednucleic acids. Certain preferred devices utilize the silicon chip orsilicon wafer formats. In preferred embodiments, the devices of thepresent invention are “microdevices”, preferably defining micromachinedstructures for use with nanoliter volumes.

[0019] The apparati, devices or chips of the invention generallycomprise a microfabricated substrate or housing defining at least afirst transport channel, or microdroplet transport channel, operablyconnected to at least a first reaction chamber, and at least a firstmeans for isothermally regulating the temperature of the reactionchamber.

[0020] The “means for isothermally regulating the temperature of thereaction chamber” may be an element, such as a particular resistor,combination of resistors, feed-back temperature detector, and/orcircuitry for temperature control, that has not been previously used inconjunction with a microfabricated device or chip for use in nucleicacid amplification. More preferably, the “means” for isothermallyregulating the temperature of the reaction chamber will be a“programmable means”. That is, a series of executable and controlledsteps, preferably in the form of a computer program, the implementationof which results in the control of the temperature of the reactionchamber within narrow limits, such that the temperature is“substantially constant”. These computer microprocessor or programmablemeans, although readily prepared by those of skill in the art, have notpreviously been proposed for use in combination with a microfabricatednucleic acid amplification device.

[0021] The microfabricated substrate of the device, chip or system isgenerally constructed so that application of a fluid in one or moretransport channels will result in the fluid being conveyed at least tothe reaction chamber. Accordingly, the microfabricated substrateinherently has a “flow-directed fabrication”. The flow-directedfabrication or construction may be based upon gravitational attraction,thermal gradients, gas or liquid pressure differences, differences inhydrophobic and hydrophilic surface structures, electrowetting, and/ordifferences in the dielectric constant between reagent fluids applied tothe substrate and the air or surrounding media. The manner in which adirectional flow capability is provided to the substrate is not criticalto the invention, so long as the substrate, device or system ultimatelyallows for the controlled manipulation of liquids or fluids appliedthereto, and effective merging and mixing where appropriate.

[0022] In the context of this invention, a “reaction” or “amplification”generally refers to reactions involving nucleic acid biomolecules, suchas RNA and DNA. “Nucleic acid amplification” generally refers to theprocess of increasing the concentration of nucleic acid, and inparticular, the concentration of a selected nucleic acid and/or adefined piece of a selected nucleic acid. “Amplified or amplificationproducts” or “amplicons” generally define the products resulting fromexecution of a nucleic acid amplification reaction.

[0023] As used herein, the term “an isothermal amplification reaction”refers to a nucleic acid amplification reaction that is conducted at asubstantially constant temperature. It will be understood that thisdefinition by no means excludes certain, preferably small, variations intemperature but is rather used to differentiate the isothermalamplification techniques from other amplification techniques known inthe art that basically rely on “cycling temperatures” in order togenerate the amplified products. Thus, the present invention isdistinguished from PCR, which fundamentally rests on the temperaturecycling phenomenon.

[0024] It will be further understood that although the isothermalamplification reactions of the present invention will generally beconducted at a substantially constant temperature, the overall executionof the amplification, diagnostic or prognostic methods of the inventionmay nonetheless require certain steps to be conducted at differenttemperatures. For example, moving fluids or microdroplets through thedifferent channels or chambers defined on the microfabricated substrate,and/or merging and mixing samples and reagents, may involve alterationsin temperature, e.g., as may be achieved via the use of defined heatingelements.

[0025] The microfabricated substrate or housing of the invention may befabricated from any one of a number of suitable materials. The materialswill preferably be of the type that can be manipulated to define thechannels, reaction chambers and other components necessary forconducting the amplification methods, and yet will be stable enough topermit repeated use in such methods once the defining components havebeen etched or otherwise imparted onto the substrate. Certain preferredexamples include, but are not limited to, silicon, quartz and glass.

[0026] The transport channels or “microdroplet transport channels”defined in the substrate are generally pathways, whether straight,curved, single, multiple, in a network, etc., through which liquids,fluids and/or gases may be passively or actively transported. Thechannels are generally etched into the silicon, quartz, glass or othersupporting substrate. The present invention requires the presence of atleast a first channel that functions to allow the transport of a fluidsample into the reaction chamber. It will be understood that such achannel need not be of a significant minimum length, and that the term“channel” therefore refers to a fluid-conveying section in functionalterms, rather than to defining a structure that is necessarily long andpipe-like.

[0027] The one or more channels in the substrate connect the variouscomponents, i.e., keep components “in communication” and moreparticularly, “in fluidic communication” and still more particularly,“in liquid communication.” Such components include, but are not limitedto, gas-intake channels and gas vents. In certain other aspects of theinvention “microdroplet transport channels” may refer to channelsconfigured (in microns) so as to accommodate “microdroplets.”

[0028] While it is not intended that the present invention be limited byprecise dimensions of the channels or precise volumes for microdroplets,illustrative ranges for channels may be between 0.5 and 50/μm in depth(preferably between 5 and 20 μm) and between 20 and 1000/μm in width(preferably 500/μm), and the volume of the microdroplets may range(calculated from their lengths) between approximately 0.01 and 100nanoliters (more typically between ten and fifty).

[0029] The first microdroplet transport channel may be operably orfunctionally connected to, or in liquid communication with, at least asecond microdroplet transport channel. First and second channels mayoperatively interact prior to connection with at least a firstisothermally regulated reaction chamber. This would be the first meaningof “connective channels”. However, other operative connections areenvisioned, and separate transport channels that function to deliverfluids to a common reaction chamber are still “interactive transportchannels” in the context of the present invention in that they conveytheir contents to a common destination.

[0030] The present invention is not limited to the number of transportchannels or other fluid-conveying means that may be provided in thesubstrate. The number and configuration of such channels will generallybe dictated by the number of reaction chambers and other componentsprovided on the substrate and/or the interaction of various individualchip elements to form a coordinated system.

[0031] At least one isothermally regulated reaction chamber is animportant element of the present invention. As used herein, an“isothermally regulated reaction chamber” is a chamber, preferably onedefining a microvolume receptacle, the temperature of which chamber maybe regulated in order to keep it substantially constant. The“substantially constant” temperature may be controlled within a fewdegrees, or within a single degree, or in certain embodiments, within afew tenths of a degree.

[0032] The means for isothermally regulating the reaction chamber mayinclude, but are not limited to, resistors in contact with or inproximity to the reaction chamber, temperature detectors, resistivetemperature detectors, dielectric sensors, or diodes and/or circuitryfor temperature control. As discussed, the isothermal regulation meanswill preferably be a programmable means. The actual means of conveyingthe heat will preferably be a sheet resistively heated (rather than awire), although polysilicon and doped polysilicon and diaphragm-typeheaters may also be used in the reaction chamber.

[0033] In certain embodiments of the present invention, themicrofabricated substrate further defines at least a first entry portoperably or functionally connected to, or in liquid communication with,at least a first microdroplet transport channel. Any one of a variety ofentry valves or ports may be used to control application of the sampleor samples.

[0034] In embodiments where the microfabricated substrate furtherdefines at least a second microdroplet transport channel, at least asecond entry port may be provided in operable or functional connection,or in liquid communication with the second microdroplet transportchannel. The invention is not limited to the number of transportchannels, nor to the number of entry ports, either in terms of ports perchannel or the total number of entry ports.

[0035] “Exit ports” or “sample collection points” are also envisioned,which are generally positioned at a downstream flow site from thereaction chamber.

[0036] In certain aspects of the invention, the microfabricatedsubstrate will further comprise a flow-directing means system in orderto facilitate that directed manipulation of fluids around the substrate.The term “flow-directing means system” is intended to refer to one ormore modifications of the substrate or other components used infunctional association with the substrate that act to control, orfurther control, the transport, merging and/or mixing of fluids ormicrodroplets between the components etched onto the underlyingsubstrate.

[0037] Certain preferred flow-directing means systems are those thatemploy a surface-tension-gradient mechanism in which discrete dropletsare differentially heated and propelled through etched channels. Aseries of heating elements may thus be arrayed along the one or moremicrodroplet transport channels. Such resistive heaters may be locatedslightly beneath the channels. In certain aspects of the invention, theheating elements are comprised of aluminum, although one or more or acombination of other suitable resistive metals or materials may beemployed, such as platinum, gold, etc.

[0038] In certain aspects of the invention, “heating element” may referto an element that is capable of at least partially liquefying ameltable material. A meltable material is “associated with” a heatingelement when it is in proximity to the heating element such that theheating element can at least partially melt the meltable material. Theproximity necessary will depend on the melting characteristics of themeltable material as well as the heating capacity of the heatingelement. The heating element may or may not be encompassed within thesame substrate as the meltable material.

[0039] Other fluid-directing means systems for use in the invention arethose that comprise a gas source in fluid communication with the one ormore transport channels and other components, such that application ofdifferential gas pressure gradients result in the controlled flow ofgases or liquids through the micromachined device.

[0040] Differences in hydrophobic and hydrophilic surface structures mayalso be employed to control the flow or transport of fluids through thedefined channels and etched components. In such embodiments, thetransport channels and/or components may comprise or may be manipulatedto comprise one or more hydrophobic regions. The channels and componentsmay also be treated with a hydrophilicity-enhancing compound orcompounds prior to addition of one or more of the biological samples oramplification reaction reagents.

[0041] “Hydrophilicity-enhancing compounds” are generally thosecompounds or preparations that enhance the hydrophilicity (wateraffinity) of a component, such as a transport channel.“Hydrophilicity-enhancing compound” is thus a functional term, ratherthan a structural definition. For example, Rain-X™ anti-fog is acommercially available reagent containing glycols and siloxanes in ethylalcohol. The fact that Rain-X™ anti-fog renders a glass or siliconsurface more hydrophilic is more important than the reagent's particularformula.

[0042] In certain aspects of the invention “hydrophobic reagents” areused to make “hydrophobic coatings” and create “hydrophobic regions”(more water repellent) in channels. It will be understood that thepresent invention is not limited to particular hydrophobic reagents. Inone embodiment, the present invention contemplates hydrophobic polymermolecules that may be grafted chemically to the silicon oxide surface.Such polymer molecules include, but are not limited to,polydimethylsiloxane. In another embodiment, the present inventioncontemplates the use of silanes to make hydrophobic coatings, includingbut not limited to halogenated silanes and alkylsilanes. The inventionis not limited to particular silanes; the selection of the silane isonly limited in a functional sense, ie., that it render the surfacehydrophobic.

[0043] In one embodiment, n-octadecyltrichlorosilane (OTS) is used as ahydrophobic reagent. In another embodiment,octadecyldimethylchlorosilane is employed. In yet another embodiment,the invention contemplates 1H, 1H, 2H, 2H-perfluorodecyltricholorosilane(FDTS, C₁₀H₄F₁₇SiCl₃) as a hydrophobic reagent. In still otherembodiments, fluoroalkyl-, aminoalkyl-, phenyl-, vinyl-, bis silylethane- and 3-methacryloxypropyltrimethoxysilane (MAOP) are contemplatedas hydrophobic reagents. Such reagents (or mixtures thereof) are usefulfor making hydrophobic coatings, and more preferably, useful for makingregions of a channel hydrophobic (as distinct from coating the entirechannel).

[0044] This invention is not limited to particular dimensions for thehydrophobic regions of the channels or components. However, while avariety of dimensions are possible, it is generally preferred that theregions have a width of between approximately 10 and 1000 μm (or greaterif desired), and more preferably between approximately 100 and 500 μm.

[0045] A surface (such as a channel surface) is “hydrophobic” when itdisplays advancing contact angles for water greater than approximately70°. In one embodiment, the treated channel surfaces of the presentinvention display advancing contact angles for water betweenapproximately 90° and approximately 130°. In another embodiment, thetreated microchannels have regions displaying advancing contact anglesfor water greater than approximately 130°.

[0046] In certain aspects of the invention, a “liquid-abuttinghydrophobic region” may refer to a hydrophobic region within a channelwhich has caused liquid (e.g., aqueous liquid) to stop or be blockedfrom further movement down the channel, said stopping or blocking beingdue to the hydrophobicity of the region, said stopped or blocked liquidpositioned immediately adjacent to said hydrophobic region.

[0047] Other flow-controlling or flow-directing means systemscontemplated for use in the present invention are those that rely on thephenomenon of electrowetting, and/or differences in the dielectricconstant between the reagent fluids and air. Electrowetting may bedescribed as the initial intake of fluid from a reservoir into achannel, electrowetting (or heating) may also be used to break thechannel droplet from contact with the reservoir. Valve sealed by amovable diaphragm and/or meltable solder can also be used to controlfluid flow.

[0048] Any one of a variety of pumps, both external and internal pumps,may be used in order to control the flow of fluids in the context ofthis invention. In certain aspects of the invention a “bubble pump” maybe used as a flow-directing means. A bubble pump operates as follows:fluid is introduced into a channel that comprises one or more electrodespositioned such that they will be in contact with a liquid sample placedin the channel. Two electrodes may be employed and a potential may beapplied between the two electrodes. At both ends of the electrodes,hydrolysis takes place and a bubble is generated. The gas bubblecontinues to grow as the electrodes continue pumping electrical chargesto the fluid. The expanded bubble creates a pressure differentialbetween the two sides of the liquid drop which eventually is largeenough to push the liquid forward and move it through the polymerchannel.

[0049] When coupled with a capillary valve, a bubble pump can actuate aneffective quantity of fluidic samples along the channel. The capillaryvalve is a narrow section of a channel. In operation, the fluidic sampleis first injected into an inlet reservoir. As soon as the fluid isloaded, it moves in the channel by capillary force. The fluid thenpasses the narrow section of the channel but stops at the edge where thechannel widens again. After the fluidic sample is loaded, a potential isapplied between two electrodes. At both ends of the electrodes,hydrolysis occurs and bubble is generated. The bubble keeps growing asthe electrodes continue pumping electrical charges to the fluid. Theexpanding bubble then creates a pressure differential between the twosides of the liquid drop, which eventually large enough to push theliquid forward.

[0050] The combination of bubble pump and capillary valve does notrequire any moving parts and is easy to fabricate. In addition, thedevice produces a well-controlled fluid motion, which depends on thebubble pressure. The bubble pressure is controlled by the amount ofcharges pumped by the electrodes. The power consumption of the device isalso minimized by this method.

[0051] In certain aspects of the invention, the flow-directing means isseparated from at least the first microdroplet transport channel by aliquid barrier. “Liquid barrier” or “moisture barrier” refers to anystructure or treatment process on existing structures that preventsshort circuits and/or damage to electronic elements (e.g., prevents thedestruction of the aluminum heating elements). In one embodiment, theliquid barrier may comprise a first silicon oxide layer, a siliconnitride layer and a second silicon oxide layer.

[0052] Further preferred aspects of the invention are those wherein themicrofabricated substrate further defines, or is operably associatedwith, a nucleic acid analysis component operably connected to or inliquid communication with the isothermally regulated reaction chamber.The operative connection between the nucleic acid analysis component andthe reaction chamber is such that the amplified nucleic acid productsgenerated by the isothermal amplification reaction can be analyzed bythe nucleic acid analysis component. The overall analytical method thusrequires that the amplified products are conveyed or otherwisetransported from the isothermally regulated reaction chamber to thenucleic acid analysis component in a manner effective to allow theirsubsequent analysis, separation, detection, or such like.

[0053] Any one of a variety of nucleic acid separation and analyticalcomponents may be used as part of the devices or systems of the presentinvention. Amplification product separation means include those for usein separation methods based upon chromatographic separation, includingadsorption, partition, ion-exchange and molecular sieve, and techniquesusing column, paper, thin-layer and gas chromatography. Gelelectrophoresis, liquid capillary electrophoresis, e.g., in glass, fusedsilica, coated and rectangular column format, polyacrylamide gel-filledcapillary columns are particularly contemplated. Gel electrophoresischannels and/or capillary gel electrophoresis channels may thus beetched into the substrate.

[0054] The use of a miniature electrophoresis stage for macromoleculeDNA separation is also contemplated. Using such a system can accomplishlarge savings of time and funds by a reduction in sample size, anincrease in processing system speed of the system, a increase in thenumber of samples handled through massive parallelism and batchfabrication techniques.

[0055] In certain embodiments, the present invention will comprises anucleic acid detection means operably connected to, or in electricalcommunication with, the nucleic acid analysis component. Visualizationmeans particularly envisioned include those using ethidium bromide/UVand radio or fluorometrically-labeled nucleotides, including antibodyand biotin bound probes. The nucleic acid detection means may thusinclude, but is not limited to, a diode detection device with suitablefilters for detection of radioactive decay, fluorescence, visible andnonvisible light wavelengths, and/or electromagnetic field changes.

[0056] The nucleic acid detection means may be a DNA sensor means, e.g.,one that detects a radiolabel or a fluorescent label. Such DNA sensormeans may be p-n-type diffusion diodes or p-n-type diffusion diodescombined with a wavelength filter and an excitation source. Siliconradiation/fluorescence detectors, photodiodes, silicon diffused diodedetectors, and other silicon fabricated radiation detectors are alsocontemplated.

[0057] The control circuitry for preferable use in the device may be “onwafer control circuitry” or “off wafer control circuitry”, the latterpreferably for use in non-glass devices. In addition to the isothermaltemperature controls, the control circuitry employed may include samplesize and flow control circuits; timing circuits; electrophoreticseparation bias, data detection and transmission control circuits; andone or more sequencer/timers to control the overall operation.

[0058] Thus the instant devices are contemplated for use in conducting adiagnostic test on a nucleic acid sample. Additionally, the presentdevices are contemplated for use in conducting a diagnostic orprognostic test on a biological sample suspected of containing aselected nucleic acid. Therefore, the present invention provides for theuse of the instant devices in the manufacture of a kit or system for theamplification of nucleic acids. In certain aspects, the inventionprovides for the use of the present devices in the manufacture of a kitor system for the diagnosis or prognosis of a disease.

[0059] Any one or more of the isothermal amplification devices or chipsof the present invention may be formulated or packaged with biologicalreagents effective to permit an isothermal nucleic acid amplificationreaction. In such aspects, the combined reagents and devices may beconsidered as “isothermal nucleic acid amplification kits”. “Biologicalreagents effective to permit an isothermal nucleic acid amplificationreaction” are exemplified by polymerases, nucleotides, buffers,solvents, nucleases, endonucleases, primers, target nucleic acidsincluding DNA and/or RNA, salts, and other suitable chemical orbiological components.

[0060] The kits may thus be defined as comprising, in suitable containermeans at least a first microfabricated substrate defining at least afirst channel, the at least a first channel connected to an isothermallyregulated reaction chamber, and reagents effective to permit anisothermal amplification reaction.

[0061] In such kits, the first microfabricated substrate may furtherdefine a nucleic acid analysis component operably connected to saidisothermally regulated reaction chamber and, optionally, a nucleic aciddetection means operably connected to the nucleic acid analysiscomponent.

[0062] The biological reagents effective for use in the amplificationreactions may be provided or packaged in any suitable form, preferablyaliquoted into suitable quantities. In certain preferred aspects, suchreagents will be provided in a dry or lyophilized formulation. Theprovision of reagents, preferably in a lyophilized form, applies to bothkits, in which the reagents are generally separately packaged, andintegral devices, in which the lyophilized reagents may bepre-fabricated into one or more etched components on the substrate.

[0063] In certain other embodiments, an effective amount of theamplifying reagents may be provided in a separate cartridge that isinterchangeably connected to the device, chip or system. Suchreplaceable cartridges or reservoirs may be provided in the same overallcontainer means as the device, chip or system or may be purchasedseparately as distinct items. Different replaceable cartridges may beprovided for conducting the various different isothermal amplificationreactions that are known in the art. A number of reagent formulationsmay be packaged together for alternative use according to the needs ofthe end user.

[0064] Diagnostic systems are also provided by the present invention,comprising at least a first microfabricated substrate defining at leasta first channel that is connected to at least a first isothermallyregulated reaction chamber; wherein the diagnostic system furthercomprises a nucleic acid analysis component and a nucleic acid detectionmeans in operable association with the reaction chamber of themicrofabricated substrate.

[0065] The diagnostic systems may also comprise, in operableassociation, at least a second microfabricated substrate defining atleast a second channel that is connected to at least a secondisothermally regulated reaction chamber. Third, fourth, fifth, tenth,20th, 50th, 100th, 500th and 1000th microfabricated substrates may alsobe provided, as is the meaning of “a plurality of microfabricatedsubstrates”.

[0066] The diagnostic systems may variously have at least a first and atleast a second microfabricated substrate, or a plurality thereof, thatare operably connected in series to a single nucleic acid analysiscomponent and nucleic acid detection means. The diagnostic systems mayalternatively comprise at least a first and at least a secondmicrofabricated substrate, or a plurality thereof, that are operablyconnected in parallel to at least two distinct nucleic acid analysiscomponents and nucleic acid detection means, or a plurality of suchcomponents.

[0067] In such kit and system embodiments, liquid handling,electrophoresis, and detector components may be coupled into anintegrated format. DNA samples may move directly between sampleprocessing, size-separation, and product detection. The components arecontrolled by electronic circuitry, fabricated on the same siliconwafer.

[0068] Accordingly, an integrated DNA sample processing design may bearrayed as multiple parallel units on a single silicon wafer. The numberof parallel DNA processing units per wafer may be maximized, andcircuitry used for overall control. A large number of simultaneousisothermal amplification reactions (up to 1000 per wafer) may beperformed on such systems.

[0069] Methods of making devices for use in isothermal nucleic acidamplification are provided by the invention, which generally comprisepreparing at least a first microfabricated device, chip or waferdefining at least a first channel that is operably connected to anisothermally regulated reaction chamber, preferably isothermallyregulated by a programmable means.

[0070] A method of making a nucleic acid diagnostic kit is alsoprovided, which generally comprises preparing at least a firstmicrofabricated device, chip or wafer defining at least a first channelthat is operably connected to an isothermally regulated reactionchamber, and combining the microfabricated device with biologicalreagents effective for use in an isothermal amplification reaction. Thecombination may be with lyophilized reagents, which may further bedisposed in the device as an integral component.

[0071] Methods of making a nucleic acid diagnostic system are furtherprovided, comprising preparing at least a first microfabricatedsubstrate defining, in a series of operable associations, at least afirst channel, an isothermally regulated reaction chamber, a nucleicacid analysis component and a nucleic acid analysis detection means.

[0072] Multi-component nucleic acid diagnostic systems may also bemanufactured by the methods of the present invention. To make amulti-component nucleic acid diagnostic system, a plurality ofmicrofabricated substrates, nucleic acid analysis and detection meansare operably combined, preferably in an interactive array or arrays.Controlling electronic circuitry and programmable regulating means arepreferably provided. Multiple parallel unit arrays on single siliconwafers are particularly preferred.

[0073] Important aspects of the present invention are methods for theisothermal amplification of selected nucleic acids or portions thereof,which methods generally comprise providing or introducing a microdropletsample comprising or suspected of comprising the selected nucleic acid,and reagents effective to permit an isothermal amplification reaction,to at least a first microfabricated substrate with an isothermallyregulated reaction chamber, as generally defined hereinabove, andconducting an isothermally regulated amplification reaction to amplifythe selected nucleic acid or a portion thereof.

[0074] As used herein, the terms “providing” or “introducing” mean thatthe sample or samples are provided or introduced into the one or moremicrofabricated substrates in a manner effective to begin theirconveyance, transportation or general movement to the isothermallyregulated reaction chamber. As described hereinabove, a number ofparticular flow-directing means systems may be employed in order toconvey the sample or samples to the reaction chamber. Where differentialheating is employed as the sole transport means, or as part of theoverall transport means, an important aspect of the invention is thatany samples that comprise enzymes for use in the isothermally regulatednucleic acid amplification reaction are “thermotransported” at atemperature below the critical temperature of the polymerase enzyme.Preferably, all samples will be transported at temperatures that arebelow the critical ranges for substantial inactivation of the enzymesfor use in the isothermal amplification reaction. It is a surprisingfeature of the invention that heat-conveying temperatures effective totransport samples into the reaction chamber can be employed that are farenough below the denaturation and/or inactivation temperatures of theenzymes necessary to catalyze the isothermal nucleic acidamplifications. The invention may thus be characterized as including amethod step of conveying said sample and/or said reagents from aninitial contact point on the microfabricated substrate to theisothermally regulated reaction chamber at a “transportingly effectivetemperature” that does not significantly denature the selectedamplification enzyme or otherwise significantly impair or reduce itscatalytic amplification activity.

[0075] The isothermal amplification reactions of the invention are alsoconducted at temperatures effective and by means effective to result inproductive mixing of the one or more samples and amplification reagents.“Effective mixing” is a functional term, most readily characterized bythe operative execution of the amplification reaction such thatamplified products may be detected. If desired, one or more samplescontaining nucleic acids and/or amplification reagents may first be“merged” prior to mixing.

[0076] In certain definitional terms, “merging” is distinct from“mixing.” When a first and second microdroplet is merged to create amerged microdroplet, the liquid may or may not be mixed.

[0077] In any event, irrespective of the degree of prior sampleassociation, the isothermal amplification reaction as a whole must beconducted under conditions effective to adequately mix the substratesand other components of the reaction. Prior to the present invention, itwas generally believed in the art that effective mixing could not beachieved at the temperatures preferred for use in the present isothermalamplification reactions. Only the endeavors of the present inventors,conducted despite the prejudices in the prior art, resulted in thediscovery that effective mixing could be achieved. Effective mixing isachievable despite the viscosity of the samples and/or reagentformulations used, and the particular biological components employed inconnection with the isothermal amplification enzyme solutions and/orsuspensions.

[0078] Those of ordinary skill in the art will be able to vary theapplication of the samples and reagents and the manner of transportingsuch components to the reaction chamber, in addition to varying theparticular details of the amplification reaction, in order to ensurethat a degree of mixing sufficient to result in amplified products isachieved. Moreover, the degree of mixing in a merged microdroplet may beenhanced by a variety of techniques provided by the present invention,including but not limited to, reversing the flow direction of the mergedmicrodroplet (as discussed herein below).

[0079] Although not in any way being limited by the following guidance,the temperature differential believed to be effective in conveyingmicrodroplet samples along a microfabricated device in accordance withthe present invention should generally be a temperature differential ofat least about 10° C. Preferably, temperature differentials of at leastabout 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C.,19° C., 20° C., about 25° C., about 30° C., about 35° C. or even up toabout 40° C. or above may be advantageously used in conveyingmicrodroplet samples along a micromachined device or substrate. It willbe understood that each of the foregoing effective conveying temperaturedifferentials must be analyzed in connection with the preferredoperating temperatures for any one or more particular amplifying enzyme,and that the temperatures chosen must be below the temperature at whichthe enzyme denatures or otherwise becomes significantly impaired in itscatalytic activity. In general, it is believed that temperaturedifferences of greater than about 30° C. will be preferred for creatingmicrodroplet motion or movement. In certain other embodiments,temperature differentials of about 40° C. will be effective, and thesetemperature gradients can be readily generated by a number of means,particularly by the use of a series of temperature sensors arrayed alongthe entire length of the one or more conveying channels etched into thesubstrate.

[0080] Although an understanding of the mechanisms of action underlyingthe surprising operability of the present invention is not necessary inorder to carry out the claimed amplification methods, the inventorsfurther point out that circulation patterns generated in the drop duringmotion aid in mixing the liquid sample. Studies using metal elements asboth heaters and temperature sensors demonstrate that a temperaturedifferential of 20-40° C. across the drop is sufficient to provideforward motion in transport channels.

[0081] Thus for only small temperature differences across the drop (onthe order of 10° C.) velocities on the order of 1 cm/s may be obtained.This velocity is more than sufficient for transporting liquid drops inMIDAT and other chip based systems.

[0082] Those of ordinary skill in the art will further understand thatother physical components of the chip fabrication will impact thetemperatures effective to transport microdroplets. By way of exampleonly, in studies using glass capillaries, it has been found that thereis a minimum temperature difference required to move the droplet. Forinstance, if the advancing angle is 36° and the receding angle is 29°(with the front of the droplet being 25° C.), then the back of thedroplet would need to be heated to ˜60° C. for a 1 mm long droplet in a20 mm high channel. This is just one example situation.

[0083] The use of channel geometry and defined chip fabrications thatnecessitate higher transport temperatures will naturally be combinedwith the use of enzymes that are functional at higher isothermalamplification temperatures. The choice of enzyme and transporttemperatures will be routine to those of ordinary skill in the art, witha number of possibilities being readily available. By way of exampleonly, methods for isothermal SDA are available in which temperatures ofbetween about 50° C. and about 70° C. are used in conjunction with athermophilic amplification enzyme. Accordingly, temperatures of about30° C., about 35° C., about 40° C., about 45° C., about 50° C., about55° C., about 60° C., about 65° C., about 70° C., or even up to about75° C. also may be employed.

[0084] However, the calculations of the present inventors indicated thatabout a 35° C. difference between the front and back of a droplet willbe sufficient to initiate droplet motion in a system with advancingangles of 36° and receding angles of 29° in a 20 mm high channel.Further studies of effective transport showed that the resultingtemperature difference was 40° C. between the front and back of thedroplet, thus corroborating the initial determination of therequirements.

[0085] This shows that the range of transporting temperatures and thevariety of enzymes for use in the invention extends to encompass each ofthe enzymes known to be suitable for use in isothermal amplifications.For example, 3SR and Qbeta-replicase are known to function at 37° C.,which can be used as part of the effective conveying temperature.Classical SDA reactions can also be conducted at a constant temperaturebetween about 37° C. and 42° C., the preferred range identified in U.S.Pat. No. 5,455,166 (incorporated herein by reference).

[0086] U.S. Pat. No. 5,455,166 is also incorporated herein by referencefor the purposes of exemplifying the level of skill in the art regardingthe selection of each component necessary for the isothermalamplification reaction. For example, this patent explains that, inaddition to the DNA polymerases, the restriction endonucleases necessaryto carry out the reaction are also mesophilic enzymes that arethermolabile at temperatures generally above the 37-42° C. advised foruse in the reaction. All such considerations will be readily employed bythose of skill in the art as they select the reagents necessary for usein the present isothermal amplification reactions.

[0087] In terms of the isothermal amplification reaction itself, ratherthan the transporting, merging and/or mixing steps, those of ordinaryskill in the art will instantly appreciate appropriate temperatures foruse in connection with the selected polymerase, replicase or otheramplification system. By way of example only, isothermal amplificationreactions involving 3SR and Qbeta-replicase may be conducted at or about37° C. Standard SDA isothermal amplification reactions may be conductedat a constant temperature between about 37° C. and 42° C. (including 38°C., 39° C., 40° C. and 41° C.), whereas isothermal SDA using athermophilic enzyme may be performed at a higher temperature range thanconventional SDA, anywhere between about 50° C. and about 70° C.

[0088] Any effective temperature that will support the desired enzymaticactivity, even if sub-optimal, may be employed in the isothermalamplification reactions of this invention. Accordingly, the isothermalamplifications may be conducted at any substantially constant andeffective temperature, including at about 20° C., 21° C., 22° C., 23°C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32°C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41°C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., and thelike.

[0089] It will be understood that the overall isothermal amplificationreaction is carried out in a manner effective to result in at leastdetectable amounts of amplified products. “At least detectable amountsof amplified products” refers to a yield of amplified nucleic acidproducts that can be detected by currently available nucleic aciddetection means. Optical methods using efficient fluorophores can detectatto-molar concentrations (corresponding to ˜10⁵ DNA molecules)migrating in capillary channels of 8×50 mm internal cross section(Woolley and Mathies, 1994; incorporated herein by reference). Reactionsfor synthesizing such DNA quantities can reasonably occur in 10 μl. Anintegrated system designed for picoliter volumes may require channeldimensions on the order of 10 μm²×100 μm (cross section×length).

[0090] In contrast to the negative beliefs in the prior art, the presentinvention has provided methods for target amplification efficiencysurprisingly equivalent to conventional SDA reactions, but conducted ona DNA chip. Amplifications of almost a million-fold have already beenachieved. This demonstrated that the physical changes in the environmenton the DNA chip, including silicon contact, temperature gradients,surface interactions and other potential inhibitors, did not adverselyaffect the amplification reaction.

[0091] In certain preferred embodiments, it is believed that theisothermal amplification reactions of the present invention will beconducted such that the sample nucleic acids are amplified at leastabout 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold,2000-fold, 5000-fold, 10,000-fold, 50,000-fold, 100,000-fold,200,000-fold, 300,000-fold, 400,000-fold, 500,000-fold, 600,000-fold,700,000-fold, 800,000-fold, 900,000-fold, or so, up to and including atleast about 1,000,000-fold, 2,000,000-fold or so.

[0092] The simplicity of sample provision to microfabricated devices isanother surprising feature of the present isothermal amplificationmethods. The samples may be provided in any “silicon-compatibleformulation”. Prior to the present invention, it was not known whetherthe various isothermal polymerases and replicases would be operative incontact with the fabricating structures of a microdevice, particularlythe preferred silicon formulations. The diligent studies of the presentinventors have shown that the present isothermal amplification methodsfunction in a “silicon-compatible manner”, and the methods of theinvention are intended to be carried out in such effective manners.

[0093] The provision of the sample to the microfabricated ormicromachined devices or systems is not believed to be critical, so longas the samples are later capable of being conveyed along the appropriatechannels. Sample sources include, but are not limited to, continuousstreams of liquid as well as static sources (such as liquid in areservoir). In a preferred embodiment, the source of liquidmicrodroplets comprises liquid in a microchannel from whichmicrodroplets of a discrete size are split off. As described above, incertain preferred embodiments, the reagents for use in the isothermalreaction will already be comprised within a pre-fabricated microdevice.In such embodiments, lyophilized reagents may be rendered active bycontact with the nucleic acid-containing sample, or alternatively, theymay be separately contacted with another fluid sample, such as a buffer.

[0094] The samples comprising the nucleic acids for application in thepresent isothermal amplification methods may be “laboratory samples” foruse in any one of a variety of molecular biological embodiments. Suchsamples may also be “biological or clinical samples”, in which case thesamples will generally be obtained from or otherwise derived from ananimal or human subject.

[0095] In any event, where the samples used are “microdroplet samples”,this term generally refers to the microdroplet themselves and samplesfrom which microdroplets may be made.

[0096] Whether the sample is a laboratory, biological or clinicalsample, the purity of the nucleic acids within the sample may varywidely. The purity of the sample is controlled only by the need to havea minimum purity necessary for successful execution of the isothermalamplification reaction. In certain embodiments, the sample will havebeen subjected to a substantial degree of extraction or purificationprior to use in the present invention, although this is not necessary inall embodiments.

[0097] In terms of the biological samples, these may be obtained from avariety of biological fluids, including blood, plasma, urine, sputum,semen, and fluids obtained from homogenized tissues. It is not believedto be necessary to limit the presence of other biological components,such as proteins and lipids, from the samples for use in the invention,although this may be desired in certain embodiments and is within thelevel of skill of the ordinary artisan.

[0098] In common with the sample preparation, the purity of thereactants provided to the device and the makeup of the device itselfrequire some degree of biocompatability in order to achieve the desiredreaction. That is to say, that the isothermal amplification reactionshould not be substantially inhibited or prevented by any componentspresent within the biological sample, contaminants within the reactantsor by the characteristics or nature of the device components, includingthe silicon fabricants.

[0099] It will be understood that the particular components, amounts ofcomponents and/or reactants and the particular conditions of thereaction may be modified in order to optimize the isothermalamplification reaction itself. All such variations and modifications areroutinely investigated in this field of study. By way of example only,one may vary the concentration of any of the components or the samples,the temperature, pH or ionic makeup of the buffers, and generally varyany other parameter of the amplification reaction.

[0100] It will be understood that the execution of the amplificationreaction, including the application of the samples and the movement,mixing and distribution of the samples prior to the actual isothermalamplification step, may also require certain optimizations. All suchvariations and optimizations will be routine to those skilled in thisfield of study.

[0101] All liquid distributions and manipulations may be performedentirely within a handling system formed as channels in micromachinedsilicon. Sensors may monitor the temperature and location of liquid inthe channels. The manipulation of reagents includes the movement,merging, mixture, and temperature control of the reagents to allownucleic acid amplification under isothermal reaction conditions.

[0102] In certain aspects of the present invention, the isothermalamplification methods and the reagents provided for use in the methodswill be based upon the strand displacement amplification reaction.Self-sustained sequence replication amplification reactions and/or Qβreplicase amplification reactions may also be used.

[0103] A preferred technique is the Strand Displacement Amplification(SDA). The SDA reaction may be conducted at a substantially constanttemperature between about 37° C. and about 42° C., or at any othereffective temperature, as exemplified herein by 52° C. It was previouslybelieved that the low temperature requirement for SDA would prevent itsuse in connection with amplification on microchip devices. However, theinventors discovered that the potential problems of stagnant temperaturegradients and reduced diffusion and sample mixing do not actually impactthe efficiency of the SDA reaction in such microvolume embodiments.

[0104] Thermophilic SDA may also be employed, as described in publishedEuropean Patent Application No. 0 684 315 (incorporated herein byreference). This technique employs thermophilic restrictionendonucleases which nick the hemimodified restriction endonucleaserecognition/cleavage site at high temperature and thermophilicpolymerases which extend from the nick and displacing the downstreamstrand in the same temperature range. At increased temperature, theamplification reaction has improved specificity and efficiency, reducednonspecific background amplification, and potentially improved yields ofamplification products.

[0105] In terms of amplified product analysis, DNA samples may besize-fractionated on an electrophoresis system built within or attachedor connected to the silicon substrate. Electrophoresed DNA products maybe visualized by radioactivity or fluorescence detectors fabricateddirectly in the silicon wafer.

[0106] In certain aspects of the invention, the amplified nucleic acidis detected by means of a detectable label incorporated into theamplified selected nucleic acid by the isothermal amplificationreaction. In other aspects, it is detected by means of a labeled probe.The label may variously be a radioisotopic, enzymatic or fluorescentlabel.

[0107] The present invention further provides methods for detecting thepresence of a selected nucleic acid, comprising introducing a samplesuspected of containing the selected nucleic acid, and reagentseffective to permit an isothermal amplification reaction, into amicrofabricated substrate defining at least a first channel, the atleast a first channel connected to an isothermally regulated reactionchamber, conducting an isothermally regulated amplification reaction toamplify the selected nucleic acid, and detecting the presence of theamplified selected nucleic acid, wherein the presence of the amplifiedselected nucleic acid confirms the presence of the selected nucleic acidin the sample.

[0108] The sample may be obtained or derived from an animal or patienthaving or suspected of having a disease. It will be understood that incertain aspects of the present diagnostic and/or prognostic methods, thepresence of the ultimate amplified selected nucleic acid will beindicative of the disease state being analyzed. In alternativeembodiments, it is the absence of amplified nucleic acid products thatis indicative of a disease state. In either embodiment, the presentinvention is ideally suited for the amplification of nucleic acids ofdefined sequence, having a defined sequence element, or including apotential point mutation, as each of the foregoing variants may bedistinguished by analyzing the amplified products resulting fromexecution of the presently claimed methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0109]FIG. 1. Example of an integrated DNA analysis system, representedschematically. The individual components of the system are injectionentry ports (A), liquid pumping channels (B), thermally (i.e.,isothermally) controlled reaction chamber (C), electrophoresis channel(D), and DNA band migration detector (E). Each component would haveassociated sensors, control circuitry, and external connections.

[0110]FIG. 2A and FIG. 2B. A two-part approach to construction of asilicon device of the present invention, and a silicon substratecomprising a plurality of devices.

[0111]FIG. 2A shows one embodiment of a single microfluidic device.

[0112]FIG. 2B shows one aspect of a silicon device comprising aplurality of microfluidic device modules.

[0113]FIG. 3A and FIG. 3B. A schematic of one embodiment of a device tosplit a nanoliter-volume liquid sample and move it using gas from a gassource.

[0114]FIG. 3A shows the liquid sample prior to splitting.

[0115]FIG. 3B shows the liquid sample after splitting off a microdropletof length L. The hatched regions represent the hydrophobic regions.

[0116]FIG. 4A and FIG. 4B. A schematic of one embodiment of a device ofthe present invention to split, move and stop microdroplets usinginternal gas pressure generation.

[0117]FIG. 4A shows a liquid sample prior to splitting.

[0118]FIG. 4B shows the liquid sample after splitting off a microdropletof length L. The hatched regions represent the hydrophobic regions.

[0119]FIG. 5. Schematic drawing showing the principle of thermallyinduced drop motion in a closed channel. The case of a single aqueousdrop in a hydrophilic channel is presented, where V is an appliedvoltage, P_(atm) is atmospheric pressure, P₂ is the receding-edgeinternal pressure, P₂ is the advancing-edge internal pressure, and θ isthe contact angle of the liquid-gas-solid interface. The contact anglewill depend on the surface characteristics of the channel and theconstituents of the drop, with a hydrophilic interaction giving θbetween 0° and 90°, and a hydrophobic surface giving θ between 90° and180°. Surface treatments can also reduce contact angle hysteresis and,therefore, reduce the temperature difference necessary for drop motion.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0120] I. Design of Microscale Devices for Isothermal AmplificationReactions

[0121] The amplification of nucleic acids provides a convenient way todiagnose a variety of disease states. However, prior to the presentinvention, it was unknown whether the movement, mixing, and merging ofviscous microvolume fluids at lower temperature to conduct isothermalamplification reactions was possible in a microfabricated environment.Isothermal amplification reactions employ reaction schemes and enzymeswhich are very different from PCR™, and it is unknown whether or not theenzymology of isothermal amplification reactions is compatible with chiphardware and materials.

[0122] Specifically, the only enzyme necessary for PCR™ amplification ofDNA targets is a thermostable DNA polymerase. Isothermal DNAamplification reactions employ additional enzymes with differentbiological activities because heat is not used to denaturedouble-stranded nucleic acids. In addition to a DNA polymerase, 3SRrequires an enzyme with RNase H activity and an RNA polymerase. The SDAreaction requires several very specific enzymatic activities which arenot necessary for PCR™ in order to successfully amplify a targetsequence. In addition to synthesizing a new DNA strand, the DNApolymerase in SDA must lack 5′-3′ exonuclease activity, either naturallyor by inactivation, incorporate the modified nucleotides required by SDA(αthio-dNTPs or other modified dNTPs), and displace a downstream singlestrand from a double stranded molecule starting at a single strandednick. In addition, the restriction endonuclease in SDA must nick (i.e.,cleave a single strand of) its double stranded recognition/cleavage sitewhen the recognition/cleavage site is hemimodified and dissociate fromits recognition/cleavage site rapidly enough to allow the polymerase tobind and amplify the target efficiently. The restriction endonucleasemust exhibit these activities under reaction conditions which arecompatible with the activities required of the polymerase.

[0123] It was not previously known if the enzymatic activities requiredfor such isothermal amplification reactions would be inhibited byinteraction with the surfaces of silicon microfabricated analysisdevices or by inhibitors present in the devices (e.g., residualchemicals from microfabrication). In addition, the change insurface-to-volume ratio which accompanies taking an enzymatic reactiondeveloped in a test tube to the microchannel of a siliconmicrofabricated device may have unpredictable effects, as changes in thediffusion properties of the reactants in the channel may interfere withthe amplification reaction. In particular for SDA, the interaction ofthe derivatized dNTPs with the microdevice environment, the effect ofthe environment on nicking activity by the restriction endonuclease andstrand-displacing activity by the polymerase were not known. It is knownthat liquid movement in a closed channel, which is a convenient meansfor bringing components of the amplification reaction into contact, isaffected by the contact angle of the liquid-gas-solid interface withinthe channel. Changes in the composition of the liquid in the channelchange the surface tension and therefore the contact angle, affectingliquid movement. The contact angle is reduced and liquid movement isfacilitated by more hydrophilic liquids such as the reaction buffersconventionally used in PCR™.

[0124] Certain isothermal amplification reactions, such as SDA, employhydrophobic components such as glycerol and BSA, which may unpredictablyaffect the surface tension properties of the liquid and the ability tomove it within the channels of microfabricated devices, particularlywhen thermocapillary pumps are used. The need to increase the amount ofheat to move the liquid aliquot with a thermocapillary pump could beincompatible with the temperature requirements of the enzymes and theisothermal amplification reaction.

[0125] Lowering the temperature of the amplification reaction may alsohave unpredictable effects. The temperature of the reaction in themicrofabricated device is typically controlled from one side of thechip, setting up a temperature gradient across the channel. Thetemperature conditions of isothermal amplification reactions would alsobe expected to alter the interactions of the reactants with the siliconor glass surfaces of the channel. Because isothermal amplification isconducted at constant, lower temperatures, the temperature gradientwhich is produced reaches equilibrium and becomes stagnant. In contrast,the temperature gradient in higher temperature reactions withthermocycling is not stagnant. Temperature fluctuations during PCR™amplification serve to minimize the gradient effect, improve diffusionof reactants and facilitate mixing.

[0126] Mixing of reactants in the channels and chambers of the DNA chipis of particular concern in isothermal amplification reactions, asmixing of reactants initiates the amplification reaction. This is notthe case in PCR™, as all reactants required for amplification arepresent together in the reaction mix. PCR™ amplification ofdouble-stranded targets does not begin until temperature cycling isstarted because until that time no single-stranded target is availableto amplify. This is not the case in isothermal amplification reactions.Because strand separation is an enzymatic process in isothermalamplification, at least one of the enzyme reactants (usually thepolymerase) is withheld until it is desired to begin the reaction. Ifthe isothermal amplification reaction starts with a heat-denaturationstep and the enzymes employed are not thermostable, all of the enzymesfor amplification are typically withheld until the target-containingsample is cooled to the appropriate reaction temperature. The samplecontaining the enzyme or enzymes must be mixed with the remainingreagents in order for amplification to begin.

[0127] To control initiation of the isothermal amplification reactionand provide an integrated nucleic acid analysis system, it is thereforehighly desirable to keep the components separate on the microfabricateddevice and bring them together to initiate amplification. This requires,however, that mixing of the two components in the channel be adequate atthe lower temperatures of isothermal amplification, and this mixing maybe negatively affected due to temperature-related decreases in diffusionand changes in surface chemistry. The components of the amplificationreaction itself may also have negative effects on mixing within thechannel. Many amplification reactions contain reagents such as glyceroland bovine serum albumin which increase viscosity and could reducemixing. The viscosity-increasing effects of these reagents is increasedat lower temperatures. It was therefore unknown whether or not therewould be adequate mixing, diffusion and temperature regulation toproduce isothermal amplification on a silicon microfabricated device.

[0128] In certain aspects, the present invention relates to movement ofmicrodroplets through microchannels, and more particularly,compositions, devices and methods to control microdroplet size andmovement. The present invention involves microfabrication of microscaledevices and reactions in microscale devices, and in particular, movementof biological samples in microdroplets through microchannels to, forexample, initiate biological reactions, particularly isothermalamplification of nucleic acids.

[0129] Although there are many formats, materials, and size scales forconstructing integrated fluidic systems, the present inventioncontemplates silicon microfabricated devices as a cost-effectivesolution.

[0130] The present invention contemplates microscale devices, comprisingmicrodroplet transport channels, reaction regions (e.g, chambers),electrophoresis modules, and radiation detectors. In a preferredembodiment, these elements are microfabricated from silicon and glasssubstrates. The various components are linked (ie., in liquidcommunication) using a surface-tension-gradient mechanism in whichdiscrete droplets are differentially heated and propelled through etchedchannels. Electronic components are fabricated on the same substratematerial, allowing sensors and controlling circuitry to be incorporatedin the same device. Since all of the components are made usingconventional photolithographic techniques, multi-component devices canbe readily assembled into complex, integrated systems.

[0131] Continuous flow liquid transport has been described using amicrofluidic device developed with silicon (Pfahler et al., 1990). Pumpshave also been described, using external forces to create flow, based onmicromachining of silicon (Van Lintel et al, 1988). The presentinvention employs discrete droplet transport in silicon using internalforces or external forces (i.e., external forces created by pumps).

[0132] As a mechanical building material, silicon has well-knownfabrication characteristics. The economic attraction of silicon devicesis that their associated micromachining technologies are, essentially,photographic reproduction techniques. In these processes, transparenttemplates or masks containing opaque designs are used to photodefineobjects on the surface of the silicon substrate. The patterns on thetemplates are generated with computer-aided design programs and candelineate structures with line-widths of less than one micron. Once atemplate is generated, it may be used almost indefinitely to produceidentical replicate structures. Consequently, even extremely complexmicromachines may be reproduced in mass quantities and at lowincremental unit cost—provided that all of the components are compatiblewith the silicon micromachining process. While other substrates, such asglass or quartz, can use photolithographic methods to constructmicrofabricated analysis devices, only silicon gives the added advantageof allowing a large variety of electronic components to be fabricatedwithin the same structure.

[0133] In one embodiment, the present invention contemplates siliconmicromachined components in an integrated analysis system, including theelements identified schematically in FIG. 1. In this proposed format,sample and reagent are injected into the device through entry ports(FIG. 1-A) and they are transported as discrete droplets throughchannels (FIG. 1-B) to a reaction chamber, such as an isothermallycontrolled reactor where mixing and reactions, such as isothermalnucleic acid amplification reactions (SDA, Qβ-replicase, etc),restriction enzyme digestion, ligation, phosphorylation,dephosphorylation, sequencing or other enzymatic or chemical reactionknown to those of skill in the art occur (FIG. 1-C). The biochemicalproducts are then moved by the same method to an electrophoresis module(FIG. 1-D) where migration data is collected by a detector (FIG. 1-E)and transmitted to a recording instrument. Importantly, the fluidic andelectronic components are designed to be fully compatible in functionand construction with the biological reactions and reagents.

[0134] A. Two-Part Approach to Construction

[0135] Most of the devices of the invention are hybrid micromechanicaldevices (two substrates bonded together). The purpose of using thismethod is to allow the fabrication of micromechanical devices out of avariety of materials (silicon, glass, fused silica, quartz, etc.). Thedevices have chamber volumes that are easily handled (sample loading,component analysis, etc.) and chamber walls that are transparent (sampleloading, fluorescent detection, etc.). The hybrid system also givesflexibility in choosing materials in one section of the unit withoutaffecting other pans of that same unit.

[0136] The invention may comprise two separate wafers of either the sameor different materials, including but, not limited to, silicon, glass,or quartz are micromachined independently. The pieces are then bondedtogether using a variety-of techniques (polyimide, UV-curing cements,anodic bonding, etc.). For transparency, a glass or quartz wafer isusually used on one side of the hybrid. In general, the sensors,heaters, and other electronic components may be patterned onto one waferand etch channels into the other. The electronic components may use 5 μmwire width over channel regions so that, if the glass wafer has theelectronic components patterned on it, the contents of the channels maybe seen.

[0137]FIG. 2A shows a two-part approach to construction. Microchannels(100) are made in the silicon substrate (200) and the structure isbonded to a glass substrate (300). The two-part channel constructiontechnique requires alignment and bonding processes but is amenable to avariety of substrates and channel profiles. In other words, formanufacturing purposes, the two-part approach allows for customizing onepiece (i.e., the silicon with channels and reaction formats) and bondingwith a standardized (non-customized) second piece, e.g., containingstandard electrical pads (400).

[0138] Hundreds or thousands of copies of a particular component can bemade simultaneously across the entire silicon wafer surface (FIG. 2B;for example, but not limited to, a wafer that is 0.5 mm thick and 100 mmin diameter). The components are made by sequential deposition, ionimplantation, or etching of thin layer materials in defined patterns.Materials that are commonly used include silicon oxide, silicon nitride,and various metals and alloys.

[0139] The technology of silicon fabrication is essentially aphotolithographic method for making machines. Once a “template” or“stencil” pattern has been prepared, additional copies of the machinesare replicated at minimal cost and effort. The density of components islimited by line-width considerations and the designing abilities of theengineers. Complete devices are made in batches and can often exceedthousands of replicates per fabrication run. Additionally, siliconfabrication has benefited from massive industrial commitment over thepast 20 years. The characteristics of the fabrication steps are knownand have been incorporated into intelligent design software orcomputer-aided design and manufacturing packages (CAD/CAM).

[0140] B. Channel Design and Construction

[0141] In silicon micromachining, a technique to form closed channelsinvolves etching an open trough on the surface of a substrate and thenbonding a second, unetched substrate over the open channel. There are awide variety of isotropic and anisotropic etch reagents, either liquidor gaseous, that can produce channels with well-defined side walls anduniform etch depths. Since the paths of the channels are defined by thephoto-process mask, the complexity of channel patterns on the device isvirtually unlimited. Controlled etching can also produce sample entryholes that pass completely through the substrate, resulting in entryports on the outside surface of the device connected to channelstructures.

[0142] The present invention contemplates a variety of silicon-based,microdroplet transport channel-containing devices. In one embodiment,the device comprises: a housing comprised of silicon, a microdroplettransport channel etched in the silicon, a microdroplet receiving meansin liquid communication with a reaction region via said transportchannels, and a liquid barrier disposed between the transport channelsand a microdroplet flow-directing means. In one embodiment, the deviceis assembled in two parts. First, the channels are etched in any numberof configurations. Second, this piece is bonded with a silicon-basedchip containing the electronics. This allows for both customization (inthe first piece) and standardization (in the second piece).

[0143] In certain aspects of the invention “conveying” may refer tocausing to be moved through, as in the case where a microdroplet isconveyed through a transport channel to a particular point, such as areaction region. Conveying may be accomplished via a flow-directingmeans.

[0144] The present invention also contemplates devices and methods forthe sealing of channels with meltable material. In one embodiment, thedevice comprises a meltable material disposed within a substrate andassociated with a heating element.

[0145] In one embodiment, the present invention contemplates a methodcomprising providing a device having a meltable material disposed withina substrate and associated with a heating element, and heating themeltable material with the heating element such that the meltablematerial at least partially liquefies and such that the substrate is notdamaged. The method may further comprise allowing the liquefied meltablematerial to cool. While the present invention is not limited by the sizeof the channel, in one embodiment the substrate further comprises amicrodroplet channel disposed in the substrate, the meltable material isdisposed within the microdroplet channel.

[0146] In another embodiment, the present invention contemplates amethod for restricting fluid flow in a channel comprising providing adevice comprising a meltable material disposed within a substrate, themeltable material associated with a heating element; and a diaphragmpositioned such that, when extended, it touches the meltable material,extending the diaphragm such that it touches the meltable material, andheating the meltable material with the heating element such that themeltable material at least partially liquefies and such that thesubstrate is not damaged. In one embodiment the method further comprisesallowing the meltable material to cool. While the present invention isnot limited by the size of the channel, in one embodiment, the substratefurther comprises a microdroplet channel disposed in the substrate, themeltable material disposed within the microdroplet channel.

[0147] In certain aspects of the invention “meltable material” may referto a material that is at least semi-solid (and preferably completelysolid) at ambient temperature, will liquefy when heated to temperaturesabove ambient temperature, and will at least partially resolidify whencooled. Preferably, meltable material at least partially liquefies at atemperature such that the substrate is undamaged. That is to say, at thetemperature the meltable material liquefies, the substrate and othermetals in the substrate does not liquefy (readily tested as set forth inExample 6) and does not change its properties. By “changing properties”it is meant that the substrate or metal maintains it structuralintegrity, does not change its conductivity and does not liquefy. Thus,the characteristic of being meltable is not necessarily associated witha particular melting point. Examples include, but are not limited to,solder, wax, polymer and plastic.

[0148] In certain aspects of the invention “solder” may refer to a metalor alloy that is a meltable material. Preferably, the solder is a lowertemperature solder, such as set forth in U.S. Pat. No. 4,967,950, hereinincorporated by reference. “Lower temperature solder” means a eutecticalloy. While the present invention is not limited to a specific solder,one preferred solder composition for the paste is a 63:37 eutectic alloyof tin:lead. Another compatible solder is a 90% metal composition havinga 63:35:2 eutectic alloy of tin:lead:silver. Other desired soldercompositions such as eutectic Pb:Sn, Pb:In, Pb:In:Sn, etc.

[0149] The present invention also contemplates a method for restrictingfluid flow in a channel, comprising providing a main channel connectedto a side channel and disposed within a substrate, meltable materialdisposed within the side channel and associated with a heating element,and a movement means connected to the side channel such that applicationof the movement means induces the meltable material to flow from theside channel into the main channel, heating the meltable material suchthat the meltable material at least partially liquefies, and applyingthe movement means such that the liquefied meltable material flows fromthe side channel into the main channel. While the present invention isnot limited by the movement means, in one embodiment the movement meansis forced air. In one embodiment the method further comprises allowingthe meltable material to cool. While the present invention is notlimited by the size of the channel, in one embodiment, the main channeland the side channel are microdroplet channels.

[0150] While the present invention is not limited by the nature of thesubstrate, in one embodiment the substrate comprises silicon or glass.Likewise, the present invention is not limited by the composition of themeltable material. In one embodiment, the meltable material comprisessolder. In a preferred embodiment, the solder comprises 40:60 Sn:Pb. Inother embodiments, the meltable material is selected from a groupconsisting of plastic, polymer and wax. Likewise, the present inventionis not limited by the placement of the meltable material in thesubstrate. In another embodiment, the meltable material is placedadjacent to a channel, while in another embodiment it is placed near thejunction of more than one channel.

[0151] II. Microfabrication of Silicon-Based Devices

[0152] As noted previously, silicon has well-known fabricationcharacteristics and associated photographic reproduction techniques. Theprincipal modem method for fabricating semiconductor integrated circuitsis the so-called planar process. The planar process relies on the uniquecharacteristics of silicon and comprises a complex sequence ofmanufacturing steps involving deposition, oxidation, photolithography,diffusion and/or ion implantation, and metallization, to fabricate a“layered” integrated circuit device in a silicon substrate. See e.g.,Miller, U.S. Pat. No. 5,091,328, hereby incorporated by reference.

[0153] For example, oxidation of a crystalline silicon substrate resultsin the formation of a layer of silicon dioxide on the substrate surface.Photolithography can then be used to selectively pattern and etch thesilicon dioxide layer to expose a portion of the underlying substrate.These openings in the silicon dioxide layer allow for the introduction(“doping”) of ions (“dopant”) into defined areas of the underlyingsilicon. The silicon dioxide acts as a mask; that is, doping only occurswhere there are openings. Careful control of the doping process and ofthe type of dopant allows for the creation of localized areas ofdifferent electrical resistivity in the silicon. The particularplacement of acceptor ion-doped (positive free hole, “p”) regions anddonor ion-doped (negative free electron, “n”) regions in large partdefines the interrelated design of the transistors, resistors,capacitors and other circuit elements on the silicon wafer. Electricalinterconnection and contact to the various p or n regions that make upthe integrated circuit is made by a deposition of a thin film ofconductive material, usually aluminum or polysilicon, thereby finalizingthe design of the integrated circuit.

[0154] Of course, the particular fabrication process and sequence usedwill depend on the desired characteristics of the device. Today, one canchoose from among a wide variety of devices and circuits to implement adesired digital or analog logic feature.

[0155] In a preferred embodiment, channels were prepared on 500 μm thickglass wafers (Dow Corning 7740) using standard aqueous-based etchprocedures. The initial glass surface was cleaned and received twolayers of electron beam evaporated metal (20 nm chromium followed by 50nm gold). Photoresist Microposit 1813 (Shipley Co.) was applied 4000rpm, 30 sec; patterned using glass mask 1 and developed. The metallayers were etched in chromium etchant (Cr-14, Cyantek Inc.,) and goldetchant (Gold Etchant TFA, Transene Co.,) until the pattern was clearlyvisible on the glass surface. The accessible glass was then etched in asolution of hydrofluoric acid and water (1:1, v/v). Etch rates wereestimated using test wafers, with the final etch typically givingchannel depths of 20 to 30 μm. For each wafer, the depth of the finishedchannel was determined using a surface profilometer. The final stripping(PRS-2000, J. T. Baker) removed both the remaining photoresist materialand the overlying metal.

[0156] In one embodiment, channels etched on glass in theabove-described manner, were bonded to the heater-element wafer in atwo-part construction approach using optical adhesive (SK-9 Lens Bond,Sumers Laboratories, Fort Washington, Pa.). The bond was cured under anultraviolet light source (365 nm) for 12 to 24 h.

[0157] Initial device design involved single layers of silicon. However,experience showed these to be inadequate to prevent short circuiting dueto (necessary) liquid microdroplets within the channels (see studiesdescribed below). The preferred design involves a triple layer ofoxides. Such a preferred device capable of moving and mixing nanoliterdroplets was constructed by bonding a planar silicon substrate tochannels etched in a glass cover. A series of metal heaters was inlaidon the silicon substrate as two parallel lanes merging into a singlelane (a “Y”-shape). The heating elements were formed by first coatingthe wafer with a 1.0 μm layer of thermal silicon dioxide. Next, 0.35 μmdeep, 5 μm wide grooves were reactive-ion etched (RIE) into the silicondioxide following the pattern set in an overlying photoresist. Aluminumwas deposited (0.35 μm) across the entire wafer using electron beamevaporation and the metal layer was “lifted-off” from all surfaceshaving intact photoresist using a stripping solution. The metal inlayprocess gives a relatively planar surface and provides a uniform basefor deposition of a solution-impermeable barrier layer. The barrierlayer is made by a sequence of three plasma-enhanced chemical vapordepositions (PECVD): 1.0 μm silicon oxide (SiO,), 0.25 μm siliconnitride (Si_(x)N_(y)) and 1.0 μm silicon oxide (SiO,). Some heatingelements were also used as resistive temperature sensors.

[0158] Heater elements were fabricated as follows. Silicon wafer(p-type, 18-22 ½-cm, boron concentration Å 10¹⁵ cm⁻³) was used as asubstrate for growth of SiO₂ thermal oxide (1 μm); photoresist(AZ-5214-E, Hoescht-Celanese) was applied and spun at 3000 rpm, 30 sec.The resist was patterned (metal 1) and developed. Reactive ion etch(RIE, PlasmaTherm, Inc.) was performed to 0.35 μm depth into the SiO₂layer at the following conditions: CHF₃, 15 sccm (standard cubiccentimeters per min); CF₄, 15 sccm; 4 mTorr; DC bias voltage of 200V,100 W, 20 min. The etch depth was measured by profilometer and 0.35 μmmetallic aluminum was electron beam deposited. The resist and overlyingmetal was lifted off by development using Microposit 11 12A remover insolution (Shipley Co.,). The barrier layers consist of sequentiallydeposited 1 μm SiOX, 0.25 μm Si_(x)N_(y), and 1 μm SiOX usingplasma-enhanced chemical vapor deposition (PECVD). RIE was used to etchcontact holes to the metal layer using a second mask (CHF₃, 15 sccm;CF₄, 15 seem; 4 mTorr; and DC bias voltage of 200V, 100 W, 120 min).

[0159] The elements are arrayed as two parallel lanes, each 500 μm wide,merging into one lane. The individual heaters consist of paired aluminumwires (5 μm) winding across the 500 μm wide region. The broad metalareas on either side of the elements are bonding locations forconnection to external circuitry. The width of the aluminum element is 5μm. The channel is uniformly etched 500 μm wide and approximately 20 μmdeep.

[0160] The heating-element wafer was bonded to a glass wafer containingetched channels with the same “Y” format. An aqueous chemical etch ofconcentrated hydrofluoric acid was used to produce channels with definedside walls and uniform depth. The etched channels are defined by achromium/gold mask and are 500 μm wide and approximately 20 μm deep. Thecomplementary silicon heater and glass channel wafers were aligned andthen bonded with adhesive to form the finished device.

[0161] Each heating element used as a temperature sensor is preferablyfirst calibrated by measurement of electrical resistance at 22° C. and65° C. under constant voltage; intermediate temperatures are estimatedby linear interpolation.

[0162] A. Microchannel Construction

[0163] There are two basic techniques that may be used for constructionof channel structures. The first technique uses a chemical or a reactiveion etch to form open channels on selected areas of a substrate. Thesechannels can range in width from 10 μm to the full thickness of thewafer (500 μm). The open channels are sealed by bonding of a secondsubstrate as a cap on top of the first one. Common bonding techniquesinclude anodic bonding, fusion bonding, melting, and epoxy bonding.Holes at specific locations for injection of the sample are then etchedfrom the backside of the cap wafer. Bonded structures have beensuccessfully applied in the implementation of capillary liquidelectrophoresis systems etched on glass substrates (Burggraf et al.,1993, ; Harrison et al., 1993). Most bonded structures are simplediscrete channel devices with a limited number of electrical componentsand interconnects. However, the bonded nature of the device means thatthe substrate material containing the electrical components may bedifferent than the cap material, adding great flexibility to devicedesign.

[0164] The second technique for the fabrication of channels relies onthe sacrificial etch technique (Mastrangelo and Muller 1989). In thistechnique, the channel is formed from a patterned thin film thatdetermines the channel height. The film is covered by the deposition ofa thick cap material and access holes are opened through it. Thesacrificial material defining the channel is next removed by chemicaletching through the access holes, and finally the channel is sealed byplugging the access holes. The main advantage of this fabricationapproach is that the channel fabrication takes place entirely on oneside of the substrate; hence this technique is referred as surfacemicromachining. The ability to pattern channels on the surface of thesubstrate brings a great deal of flexibility. Surface micromachinedchannels may be fabricated on substrates with complex topographies ofinterconnects, sensors, and control electronics. In surfacemicromachined devices, the analytical instrumentation is built alongwith the channel on the same physical substrate.

[0165] The devices by both the hybrid (bonded) and monolithic (surfacemachining) designs have been constructed. For bonded structures, bothglass and silicon substrates to form channels (500 μm wide), andt-circuits (aluminum heater circuitry, each wire filament is 5 μm wide)have been used. The monolithic device is compatible with conventionalNMOS device fabrication.

[0166] B. Channel Fabrication

[0167] The channels are made of diffused silicon on the bottom and athin film cap on the top. This type of channel may be routed throughlow-mass diaphragm-type heaters needed for the reaction. On the toplayer, a set of thin film electrodes and heaters is constructed. Boththe channels and entry port components can be formed by etching ofsilicon. The depth of etching can be controlled by prior doping of thesilicon material with an etch stop (boron).

[0168] The surface treatment of the channels may be done by immersingthe open channel in organosilane or a self-assembled monolayer coating,with oxygen reactive ion etching removing the surface from unwantedareas. Heating elements, dielectric sensors, and connecting wires may bemade from sputtered aluminum metal and conventional masking. Thesequential activation of heating elements can be computer controlledthrough external circuitry, and a printed circuit board connector.

[0169] C. Channel Treatment

[0170] Prior to performing microdroplet motion and biological reactions,the channels are preferably treated by washing with base, acid, buffer,water and a hydrophilicity-enhancing compound, followed by a relativelyhigh concentration solution of non-specific protein.“Hydrophilicity-enhancing compounds” are those compounds or preparationsthat enhance the hydrophilicity of a component, such as thehydrophilicity of a transport channel. The definition is functional,rather than structural. For example, Rain-X™ anti-fog is a commerciallyavailable reagent containing glycols and siloxanes in ethyl alcohol.However, the fact that it renders a glass or silicon surface morehydrophilic is more important than the reagent's particular formula.

[0171] “Hydrophobic reagents” are used to make “hydrophobic coatings” inchannels. It is not intended that the present invention be limited toparticular hydrophobic reagents. In one embodiment, the presentinvention contemplates hydrophobic polymer molecules that can be graftedchemically to the silicon oxide surface. Such polymer molecules include,but are not limited to, polydimethylsiloxane. In another embodiment, thepresent invention contemplates the use of silanes to make hydrophobiccoatings, including but not limited to halogenated silanes andalkylsilanes. In this regard, it is not intended that the presentinvention be limited to particular silanes; the selection of the silaneis only limited in a functional sense, ie., that it render the surfacehydrophobic.

[0172] In various aspects of the invention, n-octadecyltrichlorosilane(OTS), octadecyldimethylchlorosilane, 1H, 1H, 2H,2H-perfluorodecyltricholorosilane (FDTS, C₁₀H₄F₁₇SiCl₃), fluoroalkyl-,aminoalkyl-, phenyl-, vinyl-, bis silyl ethane- or3-methacryloxypropyltrimethoxysilane (MAOP) are contemplated ashydrophobic reagents. Such reagents (or mixtures thereof) are useful formaking hydrophobic coatings, and more preferably, useful for makingregions of a channel hydrophobic (as distinct from coating the entirechannel).

[0173] In a preferred embodiment, the channels are washed withapproximately 100 μl each of the following solutions in series: 0.1NNaOH; 0.1N HCl; 10 mM Tris-HCl (pH 8.0), deionized, H₂O, Rain-X Anti-Fog(a hydrophilicity-enhancing compound commercially available from UnelkoCorp., Scottsdale, Ariz), and 500 μg/μl bovine serum albumin(non-specific protein commercially available in restriction enzyme gradefrom GIBCO-BRL). The wafer was placed on a stereoscope stage (OlympusSZ1145), and the contact pads for the heating elements were connected toa regulated power supply. Heating occurred by passing approximately 30volts through the element in short pulses and observing the movementrate of the droplets. A detectable reduction in droplet volume fromevaporation was noted in each study, usually of less than 30%. Dropletmovement was recorded with a Hamamatsu video camera on videotape.

[0174] It is not intended that the present invention be limited toparticular dimensions for the hydrophobic regions of the presentinvention. While a variety of dimensions are possible, it is generallypreferred that the regions have a width of between approximately 10 and1000 μm (or greater if desired), and more preferably betweenapproximately 100 and 500 μm.

[0175] A surface (such as a channel surface) is “hydrophobic” when itdisplays advancing contact angles for water greater than approximatelyseventy degrees. In one embodiment, the treated channel surfaces of thepresent invention display advancing contact angles for water betweenapproximately ninety (90) and approximately one hundred and thirty (130)degrees. In another embodiment, the treated microchannels have regionsdisplaying advancing contact angles for water greater than approximatelyone hundred and thirty (130) degrees.

[0176] D. Glass Channel and Chamber Fabrication

[0177] The channel and the chamber fabrication begins by depositing 0.4μm metallic layer of Gold (Electron beam deposition) on the surface of500 μm thick glass water (Dow Coming 7740). A 0.06 μm layer of chromiumis used as the adhesion layer. Photoresist is applied and patternedusing glass mask 1 and developed. The metal layers are etched in goldetchant (Gold Etchant TFA, Transene Co.) and Chromium etchant (CR-14,Cyantec Inc.). The accessible glass is then etched in a solution offreshly prepared hydrofluoric and nitric acid (7:3, v/v). The etch rateis approximately 5 μm/min and the etch depth is conveniently measuredusing a surface profilometer. The metal layers are removed and the waferrinsed in DI water, air dried and oven dried at 100° C. for 20 min. Thefollowing processing steps are done for patterning hydrophobic regionsonto the glass surface.

[0178] 1. Hydrophobic Patterning of Glass Substrate

[0179] A 1.5 μm thick aluminum layer was electron beam deposited,covering the etched channels and chamber. A thick photoresist (AZ 4620)is applied and spun at 750 rpm for 50 sec . The resist is patterned (SAMMask) and developed. The exposed aluminum is etched in aluminum etchant.The photoresist is stripped off in hot PRS 2000 (J. T. Baker). Thesamples are then cleaned in acetone, isopropyl alcohol and DI water for5 min each and the water dried off in a 100° C. oven of 10-15 min. Thesamples are then dipped in a 1% OTS solution in toluene for 10-15 min.The SAM deposition was carried out in a chemical hood. The samples werethen rinsed in toluene, isopropyl alcohol and DI water for 5 min each.Next, they were put in aluminum etchant until all metallic aluminum wasremoved. The samples were then rinsed in DI water and air dried. For thedevices with the inlet from the top, holes were drilled byelectrochemical discharge drilling.

[0180] The glass side was then aligned on top of the silicon side andthen bonded together using optical adhesive (SK-9 Lens Bond, SumersLaboratories, Fort Washington, PA). The bond was cured under anultraviolet light source (365 nm) for 24 h.

[0181] E. Heaters and Resistive Temperature Detectors

[0182] The fabrication process for the heater and temperature detectorbegins by using Silicon water (p-type, 18-22 alun-cm, boronconcentration ˜10¹⁵ cm³) as a substrate for growth of S102 thermal oxide(1 μm). A 0.5 μm metallic Aluminum film is electron beam deposited.Photoresist PR 1827 is applied and spun at 4000 rpm for 30 sec,patterned (metal 1) and developed. The exposed aluminum is etched inaluminum etchant and the photoresist stripped to define the metalheater.

[0183] Photoresist is spun again and a second lithography is done (metal2). A 0.15 μm layer of platinum (“Pt”) is electron beam deposited. A0.03 μm thick titanium metal layer (electron beam deposited) is used asthe adhesion layer. The resist and the overlying metal is lifted off bydevelopment using Microposit 1112A remover in solution (Shipley Co.).This platinum metal will be used as the resistive thermal detector.Next, 0.7 μm of low temperature oxide (LTO) of silicon is deposited toact as the barrier layer and the hydrophilic substrate. A thirdlithography is done and the LTO is etched in buffered hydrofluoric acidto open contacts to the metal contact pads. The further processing stepsare done to pattern hydrophobic regions onto the hydrophilic siliconoxide surface.

[0184] 1. Hydrophobic Patterning of Silicon Oxide Substrate

[0185] A 0.1 μm layer of chromium metal is electronbeam deposited on theprocessed water. Photoresist PR 1827 is applied and spun at 2000 rpm for30 sec. The resist is patterned (SAM mask) and developed. The exposedchromium metal is etched in chromium etchant to expose the silicon oxideand the photoresist is then stripped off. The samples are then cleanedin acetone, isopropyl alcohol and DI water for 10 min each, air driedand oven dried at 100° C. for 5 min. The samples are then put in 1 wt %octadecyltrichlorosilane (OTS) solution in toluene for 15-30 min. OTSdeposits on the samples as a self assembled monolayer (SAM). The samplesare then rinsed in toluene, isopropyl alcohol and DI water for 5 mineach, and then oven dried (100° C., 5 min). Next, they are put inchromium etchant to remove the chromium layer below. The SAM on thechromium metal gets lifted off as a result of this. The samples werethen rinsed in DI water and air dried, resulting in regions of intacthydrophobic regions on a hydrophilic oxide substrate. Heater elementsand RTDs have also been fabricated on a quartz substrate. Thefabrication steps are similar to that of the silicon processing steps.

[0186] Once the appropriate chemicals are added to the DNA sample, thesolution may be passed through several different temperatures. The mixedsolution may be transported to a uniformly heated reaction chamber ofthe unit. Once in the chamber, the temperature of the solution may beincreased using local heaters and temperature sensors. The temperatureof the ends of the drops may be monitored and maintained at the sametemperature to prevent the drop from leaving the reaction zone. If thedrop does begin to move, local temperature gradients could quicklystabilize the drop. The cooling of the drop may be accomplished bysimple conduction of the heat through the walls of the channel toambient temperature.

[0187] F. Fluid Mixing Chamber

[0188] The mixing chamber consists of an enlarged portion of themicrochannel structure, with one or more microchannels connected to thechamber. The mixing chamber is suspended on a thin silicon nitridediaphragm. This construction allows for excellent thermal isolation, asneeded for low power heat cycling of the mixture. Construction ofmembrane suspended structures has been demonstrated (Mastrangelo et al.,1991). The heating is effected with a set of concentric resistors(heaters) that are placed on the periphery of the mixing chamber. Thisdesign, along with the high thermal conductivity of the liquid sample,makes the chamber temperature quite uniform. Along with the heaters,temperature sensors (diodes) are constructed on the diaphragm to monitorthe temperature of the mixture. The low mass construction of the chamberallows for rapid heating cycles. Temperature control may handle samplesof variable volume and heat capacity. The chamber also contains a set ofelectrodes and heating elements to drive the mixture out of the chamberat the completion of the reaction.

[0189] G. Electrophoresis and Detector Component Design

[0190] The present invention contemplates one or more gelelectrophoresis modules as a component of the microscale device.Reducing the thickness of the electrophoresis channel may improveresolution. Thinner gels dissipate heat more readily and allow highervoltages to be used, with concomitant improvements in separation. Theposition and width of the electrophoresis detector are also critical tothe ultimate resolution of the electrophoresis system. A micromachinedelectronic detector, such as a photodiode, placed in the underlyingsilicon substrate may be less than one micron from the gel matrix andcan have a width of 5 microns or less. Since the gel length required forthe resolution of two migrating bands is proportional to the resolutionof the detector, the incorporation of micron-width electronic detectorscan reduce the total gel length required for standard genotyping by atleast an order of magnitude.

[0191] To demonstrate that standard gel electrophoresis can operate inmicron-diameter channels, modules were fabricated using etched glasschannels and fluorescent-labeled DNA (YOYO intercalating dye).Polyacrylamide gel electrophoresis of a complex DNA mixture wasperformed in a channel 500 μm wide and 20 μm deep. The electrophoresiswas performed with the positive electrode to the right and the DNAsample applied at the left. The DNA sample (Bluescript KS digested withMspI) is labeled with intercalating UV-fluorescent dye (YOYO-1) and isvisualized under incandescent light. Separation of the component bandsis clearly visible less than 300/μm from the buffer reservoir-to-gelinterface. The high resolution of the detector (in this case, amicroscope) allowed the use of an unusually short gel, resolving severalclosely eluting bands.

[0192] H. Miniature Electrophoresis Chamber

[0193] A 20 μm×500 μm×4 cm channel etched into a glass wafer was used asan electrophoresis chamber. The channels may be made by three differentprocesses: a glass channel wet-etched, a silicon channel dry-etched(RIE), or a silicon channel wet-etched. Although the edges of thechannel are rough and the walls of the channels are not vertical, thefloor of the channel is quite smooth. Better channels may also beconstructed with silicon as the base material using dry or wet etching.The glass channel was then bonded to a quartz slide using UV-cure cementand loaded with a 15% polyacrylamide gel and 1× TBE running buffer. Thegel was loaded with DNA ladder (BSKSIMSPI 50-500 bp), stained with TOTOfluorescent dye, and placed in a ˜3 volt/cm field for 30 minutes. Atthese short times and low voltages, separation into visibly resolvedbands is obtained.

[0194] I. Integrated Electrophoresis/Detection Device

[0195] Monolithic devices created from silicon have the advantage thatno bonding is necessary and that electronic components may be integratedwith the mechanical system in any location. A silicon micromachined gelelectrophoresis channel integrated with a silicon radiation/fluorescencedetector underneath it was fabricated. The “die” measures 1.25×1.25 cmand contains about 20 different types of gel devices. Among thesedevices, the channel width varies from 20 to 150 μm, and the channelheight is approximately 3 μm. There are different channel formatsincluding straight, folded, and looped channels, each of which has atleast one DNA detector. The longest channel on this wafer is a 9.5 cmlong folded channel. For folded channels, as long as the channel bendsare paired curves, it may be shown that the electric field is uniformaround the bend and the solute bands start and end as uniform bands.

[0196] The structure primarily comprising a silicon diffused diodedetector (Kemmer, 1980; Wouters and van Sprakelaar 1993) fabricatedunderneath a gel channel. The diode is fabricated on a high purityp-type float zone substrate to assure a good carrier lifetime. A layerof silicon dioxide is used as a passivation layer below a siliconnitride blocking layer. The electrodes for the electrophoresis stage areformed by deposition and patterning of n+polycrystalline silicon. Thechannel for the microgel is built with two layers of phosphoslicateglass as described in Mastrangelo and Muller 1989; Mastrangelo andMuller 1989. The cap of the channel is deposited using a thin siliconnitride dielectric and a 2 m-thick undoped polysilicon shell. A seriesof etching holes are patterned on the side or top of the shell down tothe phosphosilicate glass and used to sacrificially etch thephosphosilicate glass (Mastrangelo and Muller, 1989) thus forming thechannel cavity. The cavity can then be refilled with polyacrylamide gel.

[0197] The invention has tested the radiation/fluorescence detectors andperformed simple DNA separations with them. The experiments wereperformed using a P³² labeled DNA source placed on top of the detector.Note that the chip used for this test did not have the channels formedon the surface and contained as an insulating layer. Pulse-shaped(Knoll, 1989) scope traces or the measured signals from the diodedetector were detected from sample DNA. Not only is the response rapid(≈1 μs), but a single decay event (each trace is from only one particle)may be detected.

[0198] Fluorescent DNA may also be detected with the same detector. Adetector was mounted in a 24 pin IC package and covered with a SYBRgreen gel filter (the filter was ˜1 cm from the detector surface). Aglass slide was placed over the filter and ˜40 μl of 0.03 μg/μg of SYBRgreen labeled DNA solution was placed on the slide (contained by silicongrease wells). The sample was illuminated using a Ziess Axioskop UVsource with a ˜490 nm filter. The reverse current was measured with anHP 4145B semiconductor parameter analyzer as a function of the biasvoltage. The signal from the SYBR sample is approximately twice thecontrol signal (DI water). Although this experiment was not performedunder optimum conditions, it clearly demonstrates that the detector iscapable of detecting fluorescent DNA.

[0199] Sample separation experiments have also been performed using thisdetector. A 100 μm ID capillary tube filled with 10% polyacrylamide wasglued on top of a detector (same as that described above) approximately1.5 cm from the sample injection end. A 100 bp and a 300 bp PCR™ product(50/50 mixture) was electrokinetically injected into the channel forapproximately 5 min using a field of 25 V/cm after which the sample wellwas flushed and refilled with running buffer. The results of the 125minute run show the detection of the radioactive primers and the twoPCR™ products. Note that, although the radiation detection scheme maynot be used in the final sequencing system, it is very useful toevaluate the electrophoresis chambers until the necessary, fluorescentfilters are constructed and tested.

[0200] The present invention contemplates an electrophoresis unit thatintegrates a micromachined channel and an electronic DNA detector. Thechannel is constructed using a sacrificial etch process on a singlesilicon wafer rather than the bonded surface-etch method describedearlier. In the sacrificial etch technique, the channel configuration ispatterned by depositing on the wafer surface an etch-sensitive material(phosphosilicate glass, SiO₂.P_(x)) with a thickness equivalent to thedesired channel height. A triple-layer overlay of plasma-enhancedchemical vapor deposited silicon nitride, undoped polycrystallinesilicon, and silicon nitride (Si_(x)N_(y)/polySi/Si_(x)N_(y)) completelycovers the sacrificial material with the exception of small access holeson the top or sides. A selective liquid etch removes the sacrificiallayer material, but not the overlay or the underlying substrate. Thesacrificial etch technique results in a complete channel being formeddirectly on the substrate containing the electronic components. The 3 μmdeep channel has two buffer reservoirs on either end with integralphosphorus-doped polycrystalline silicon electrodes. The channel heightformed by this technique (˜3/μm) is considerably smaller than the heightof the bonded structures due to the limitations of the sacrificial layerdeposition and the strength of the overlying layer. Note that, for thesechannel dimensions, liquid drops would have volumes on the order ofpicoliters.

[0201] The diffusion regions of the doped-diffusion diode radiationdetector elements fabricated on a silicon wafer are approximately 300 μmlong and 4 μm wide, and are flanked by the guard ring shieldingelectrodes.

[0202] A radiation detector, consisting of a 10 μm wide “p-n”-type diodewith a 5 μm wide guard ring around the outer edge, is fashioned directlyinto the silicon substrate underneath the channel. In thisimplementation, an integral radiation detector was chosen because ofhigh sensitivity (a single decay event), small aperture dimensions, andwell-know fabrication and response characteristics. On thiselectrophoresis system, a 1 cm long, 3 μm thick gel is able to performas separation on a 80 and a 300 base-pair fragment of DNA. It should benoted that this diode, although currently configured for high-energybeta particle detection, can also operate as a photon detector. Withproper wavelength filters and light sources, detection of fluorescenceemission may be accommodated with a similar device.

[0203] Radiation detectors were prepared as follows. A 200 ½-cm, floatzone, boron-doped, p-type silicon wafer was used as a substrate.Diffused layers of phosphorus (5×10⁴ cm⁻²) and boron (1×10¹⁵ cm⁻²) wereion-implanted onto the sample in lithographically-defined regions;thermal silicon oxide was grown (0.2 μm at 900° C.) over the wafer; andcontact holes were etched to the diffusion layer using bufferedhydrofluoric acid solution (5:1). A 3.3 μm layer of Microposit 1400-37photoresist was patterned to define the metal pads; 50 nm chromiumfollowed by 400 nm gold was evaporated over the resist; and themetallization lifted off the regions retaining the resist. A layer ofMicroposit 1813 photoresist was applied across the wafer and baked for110° C. for 30 min to form an aqueous solution barrier. Radioactivephosphorus (³²P) decay events could be detected using a sample oflabeled DNA in PCR™ reaction buffer placed on the photoresist layer. Thedetector was connected to a charge-sensitive preamplifier (EV-Products550A), followed by a linear shaping amplifier and a standardoscilloscope.

[0204] An oscilloscope trace of output from the radiation detectorshowing individual decay events from ³²P-labeled DNA was generated afterthe aqueous DNA sample was placed directly on the detector and sampledfor 30 sec. The screen is displaying a vertical scale of 0.5V/divisionand horizontal scale of 20 μsec/division.

[0205] J. Gel Voltage and Temperature Control Circuits

[0206] The control circuitry and software for the integrated DNA sampleprocessing and sequencing devices are a further aspect of the invention.In particular, the devices will require circuitry for signal bufferingand for the multiplexing of control signals. A microprocessor, eitherexternal or on-wafer, determines the synchronization of events on thedevice and store the output information.

[0207] Temperature control of gel occurs by heating with polysilicon orthin metal resistors imbedded in the surface of the wafer immediatelybeneath the channel. The precise temperature control of the gel isrequired as minute fluctuations contribute to the dispersion of themigrating sample and non-uniform bands. The power distribution andoptimal heater placement is determined for each electrophoresis designby solving the relevant heat transfer equations. As long as the walls ofthe electrophoresis channel are maintained at the appropriatetemperature and the height of the channel is constructed uniformly, theinternal temperature of the across the gel should not vary by more than1.0° C. and be maintained at any arbitrary temperature.

[0208] Although the electrophoresis voltages may be low, the potentialuse of high voltages in the gel electrophoresis channels willnecessitate care in fabricating the silicon oxide/siliconnitride/silicon oxide insulating layer. Silicon nitride and siliconoxide have a breakdown field voltage of about 200-1000 V/μm (Sze, 1967;Harari, 1977; Sze, 1981). Consequently, the layers between the siliconcircuitry (including the diode detectors) and the electrically activegel are approximately 2 to 4 microns thick. The possible presence ofminute “pinholes” in the LPCVD deposited layers must also be carefullymonitored, since such holes can provide local weak points in theinsulation of the silicon circuitry. However, the routine use of siliconnitride as a mask for wet etch processes in solid-state fabricationindicate that pinholes are insignificant.

[0209] Glass may be used as their substrate material. In a glass-baseddevice, any associated on-wafer circuitry must be constructed onpolysilicon thin films adjacent to the electrophoresis channels (Tickle,1969). As an alternative are designs that energize small fractions ofthe channel at a time, thereby decreasing the voltage required withoutsacrificing resolution. Cyclical or “loop” 9.5 cm channels wereconstructed to test this (Sun and Hartwick, 1994). However, since activeelectrodes are in immediate contact with the get matrix care must beexercised so as not to irreversibly adsorb the DNA samples on theelectrodes. Alternative gel channel designs are possible.

[0210] In one embodiment of the device of the present invention, thedevice comprises a glass top bonded to a silicon substrate containingthe heater, the contact pad and the resistive temperature detector. Theglass side has channels and chambers etched into it. Inlet and overflowports, a gas vent and a air chamber are also part of this embodiment.

[0211] III. Fluid Movement

[0212] The present invention contemplates a method for movingmicrodroplets, comprising providing a liquid microdroplet disposedwithin a microdroplet transport channel etched in silicon, the channelin liquid communication with a reaction region via the transport channeland separated from a microdroplet flow-directing means by a liquidbarrier, and conveying the microdroplet in the transport channel to thereaction region via the microdroplet flow-directing means. It isintended that the present invention be limited by the particular natureof the microdroplet flow-directing means. In one embodiment, itcomprises a series of aluminum heating elements arrayed along thetransport channel and the microdroplets are conveyed by differentialheating of the microdroplet by the heating elements.

[0213] It has been found empirically that the methods and devices of thepresent invention may be used with success when, prior to the conveyingdescribed above the transport channel (or channels) is treated with ahydrophilicity-enhancing compound. It is not intended that the inventionbe limited by exactly when the treatment takes place. Indeed, there issome flexibility because of the long-life characteristics of someenhancing compounds.

[0214] It has also been found empirically that the methods and devicesof the present invention may be used with success when regions of themicrochannel are treated with hydrophobic reagents to create hydrophobicregions. By using defined, hydrophobic regions at definite locations inmicrochannels and using a pressure source, one can split off precisenanoliter volume liquid drops (i.e., microdroplets) and control themotion of those drops though the microchannels.

[0215] In one embodiment employing such hydrophobic regions (or“hydrophobic patches”), the present invention contemplates a method formoving microdroplets, comprising providing microdroplet transportchannel (or a device comprising a microdroplet transport channel), thechannel having one or more hydrophobic regions and in communication witha gas source; introducing liquid into the channel under conditions suchthat the liquid stops at one of the hydrophobic regions so as to definea source of liquid microdroplets disposed within the channel and aliquid-abutting hydrophobic region, and separating a discrete amount ofliquid from the source of liquid microdroplets using gas from the gassource under conditions such that a microdroplet of defined size comesin contact with, and moves over, the liquid-abutting hydrophobic region.

[0216] In one embodiment, the gas from the gas source enters the channelfrom a gas-intake pathway in communication with the microdroplettransport channel and exits the channel from a gas vent that is also incommunication with the microdroplet transport channel. It is preferred,in this embodiment, that the introduction of liquid into the channel (asset forth in the above-described method) is such that the liquid passesover the gas-intake pathway and the desired size of the microdroplet isdefined by the distance between the gas-intake pathway and theliquid-abutting hydrophobic region. In this embodiment, introduction ofthe gas (as set forth in the above-described method) forces themicrodroplet to pass over the liquid-abutting hydrophobic region andpass by (but not enter) the gas vent.

[0217] In another embodiment employing such hydrophobic regions (or“hydrophobic patches”), the present invention contemplates a method formoving microdroplets, comprising: providing a device comprising amicrodroplet transport channel etched in silicon, the channel having oneor more hydrophobic regions and in communication with a gas source;introducing liquid into the channel under conditions such that theliquid stops at one of the hydrophobic regions so as to define a sourceof liquid microdroplets disposed within the channel and a liquidabutting hydrophobic region, and separating a discrete amount of liquidfrom the source of liquid microdroplets using gas from the gas sourceunder conditions such that a microdroplet of defined size comes incontact with, and moves over, the liquid-abutting hydrophobic region.

[0218] Again, it has been found empirically that there is a need for aliquid barrier between the liquid in the channels and the electronics ofthe silicon chip. A preferred barrier comprises a first silicon oxidelayer, a silicon nitride layer, and a second silicon oxide layer.

[0219] The present invention further contemplates a method for mergingmicrodroplets comprising providing first and second liquidmicrodroplets, a liquid microdroplet delivering means, and a device,said device comprising a housing comprised of silicon, first and secondmicrodroplet transport channels etched in the silicon and connecting toform a third transport channel containing a reaction region, amicrodroplet receiving means in liquid communication with the reactionregion via the transport channels, and microdroplet flow-directing meansarrayed along the first, second and third transport channels deliveringthe first liquid microdroplet via the microdroplet delivering means tothe first transport channel, delivering the second liquid microdropletvia the microdroplet delivering means to the second transport channel,and conveying the microdroplets in the transport channels to thereaction region in the third transport channel via the microdropletflow-directing means, thereby merging the first and second microdropletsto create a merged microdroplet.

[0220] In one embodiment, said first microdroplet comprises nucleic acidand the second microdroplet comprises a nuclease capable of acting onthe nucleic acid. In this embodiment, it is desirable to enhance themixing within the merged microdroplet. This may be achieved a number ofways. In one embodiment for mixing, after the conveying of step, theflow direction is reversed. It is not intended that the presentinvention be limited by the nature or number of reversals. If the flowdirection of the merged microdroplet is reversed even a single time,this process increases the mixing of the reactants.

[0221] The present invention contemplates methods, compositions anddevices for the creation of microdroplets of discrete (i.e., controlledand predetermined) size. The present invention contemplates the use ofselective hydrophobic coatings to develop a liquid-sample injection andmotion system that does not require the use of valves. In oneembodiment, the present invention contemplates a method of lift-off topattern hydrophobic and hydrophilic regions on glass, quartz and siliconsubstrates, involving the deposition of a hydrophobic reagent (such as aself-assembled monolayer film of OTS) on a silicon oxide surfacepattered by a metal layer and subsequent removal of the metal to givehydrophobic patterns. Other substrates such as plastics may also be usedafter depositing a think film of silicon oxide or spin-on-glass.

[0222] Previous work in patterning hydrophobic surfaces have been doneby photocleaving of such monolayer films. The photocleaving procedureuses Deep-UV exposure to make the molecules of the monolayerhydrophilic. By contrast, the present invention contemplates a methodwhich eliminates the use of high-power UV source; rather the preferredmethod of the present invention uses microfabrication procedures.

[0223] Following the proper hydrophobic patterning of the surface (e.g.,the surface of a microdroplet transport channel), the present inventioncontemplates the placement of a patterned etched glass cap over thepattern on a flat surface. The hydrophobic/hydrophilic channels thusformed can then be used to move precise nanoliter-volume liquid samples.

[0224]FIG. 3A and FIG. 3B show a schematic of one embodiment of a device(10) to split a nanoliter-volume liquid sample and move it usingexternal air, said device having a plurality of hydrophobic regions(hatched regions). Looking at FIG. 3A, liquid (shown as solid black)placed at the inlet (20) is drawn in by surface forces and stops in thechannel at the liquid-abutting hydrophobic region (40), with overflowhandled by an overflow channel and overflow outlet (30). In theembodiment shown in FIG. 3A, the from of the liquid moves by (but doesnot enter) a gas-intake pathway (50) that is in fluidic communicationwith the channel; the liquid-abutting hydrophobic region (40) causes theliquid to move to a definite location. Gas from a gas source (e.g., airfrom an external air source and/or pump) can then be injected (FIG. 3B,lower arrow) to split a microdroplet of length “L”. The volume of themicrodroplet split-off (60) is pre-determined and depends on the length“L” and the channel cross-section. To prevent the pressure of the gas(e.g., air) from acting towards the inlet side, the inlet (20) andoverflow ports (30) may be blocked or may be loaded with excess water toincrease the resistance to flow.

[0225] The patterned surfaces may also be used to control the motion ofthe drop. By placing a hydrophobic gas vent (70) further down thechannel, one can stop the liquid microdroplet (60) after moving beyondthe vent (70). As the drop (60) passes the vent (70), the air will goout through the vent (70) and will not push the drop further.

[0226] One can start moving the drop (60) again by blocking the vent(70). By using a combination of hydrophobic air pressure lines,hydrophobic vents and strategic opening and/or closing of vents, one canmove the liquid drop back and forth for mixing or move it to preciselocations in a channel network to perform operations such as heating,reaction and/or separations.

[0227] In addition to using external air, one can also use internallygenerated air pressure to split and move drops. FIG. 4A and FIG. 4B showa schematic of one embodiment of a device (110) of the present inventionto split (e.g., define), move and stop microdroplets using internal gas(e.g., air) pressure generation, said device having a plurality ofhydrophobic regions (hatched regions). Looking at FIG. 4A, liquid (shownas solid black) placed at the inlet (120) is drawn in by surface forcesand stops in the channel at the liquid-abutting hydrophobic region(140), with overflow handled by an overflow channel and overflow outlet(130). In the embodiment shown in FIG. 4A, the front of the liquid movesby (but does not enter) a gas-intake pathway (150) that is in fluidiccommunication with the channel. By heating air trapped inside chambers(180) that are in fluidic communication with the microdroplet transportchannel via the gas-intake pathway (150), an increased pressure may begenerated. The magnitude of the pressure increase inside a chamber ofvolume V is related to the increase in temperature and may be estimatedby the Ideal Gas relation.

[0228] Increasing the temperature of the gas (e.g., air) will cause thepressure inside the chamber to rise until the pressure is high enough tosplit off a drop (160) and move it beyond the liquid-abuttinghydrophobic region (140). In order to avoid the problem of the expandedair heating up the liquid, the chamber may be placed at a distance fromthe transport channel. Moreover, having the heaters suspended inside theair chamber or placing them on a thin insulation membrane will not onlyavoid cross-talk, but will involve a minimal power consumption.

[0229] The compositions and methods are suitable for devices having avariety of designs and dimensions, including, but not limited to,devices with chamber volumes from 0.24 mm³to 0.8 mm³ for channeldimensions of 40 μm by 500 μm. Drop splitting and motion is seen with1-3 sec using voltages between 4.5 volts to 7.5 volts (the resistance ofthe heaters varied between 9.5 ohms to 11 ohms). The size of the dropsplit is between approximately 25 and approximately 50 nanoliters,depending on the value “L” used for the channel design. Keeping theheaters actuated keeps the microdroplet moving almost to the end of thechannel (a distance of around 125 mm); the time taken depends on thevoltage applied to the heater and the volume of the chamber. Initiationof drop motion is seen sooner for the operation of devices with smallerchambers. While an understanding of precise mechanisms is not needed forthe successful practice of the present invention, it is believed thatwith smaller chamber, the volume is smaller and higher values ofpressure are achieved more quickly. The maximum temperatures reachednear the heater are approximately 70° C. measured by the RTD.

[0230] A. Movement of Discrete MicroDroplets

[0231] The present invention contemplates microscale devices, comprisingmicrodroplet transport channels having hydrophilic and hydrophobicregions, reaction chambers, gas-intake pathways and vents,electrophoresis modules, and detectors, including but not limited toradiation detectors. In some embodiments, the devices further compriseair chambers to internally generate air pressure to split and movemicrodroplets (i.e., “on-chip” pressure generation).

[0232] The present invention describes the controlled movement of liquidsamples in discrete droplets in silicon. Discrete droplet transportinvolves a system using enclosed channels or tubes to transport theliquid to the desired locations (FIG. 1-B). Within the channels,discrete liquid reagent microdroplets may be injected, measured, andmoved between the biochemical analysis components. Discrete dropletmovement has three advantages. First, each sample droplet is separatedfrom all others so that the risk of contamination is reduced. Second, ina uniform channel, the volume of each sample may be determined by merelymeasuring the droplet length. Third, the motion of these droplets may beaccomplished with heating (i.e., using internal forces and no movingparts). Movement is performed using thermal gradients to change theinterfacial tension at the front or back of the droplets and, thus,generate pressure differences across the droplet. For example, a dropletin a hydrophilic channel may be propelled forward by heating the backinterface. The local increase in temperature reduces the surface tensionon the back surface of the droplet and, therefore, decreases theinterfacial pressure difference. The decreased pressure differencecorresponds to an increase in the local internal pressure on that end ofthe droplet (PI increases). The two droplet interfaces are no longer inequilibrium, with P₁ greater than P₂, and the pressure differencepropels the droplet forward.

[0233] That is to say, forward motion may be maintained by continuing toheat the droplet at the rear surface with successive heaters along thechannel, while heating the front surface may be used to reverse themotion of the droplet. Applying a voltage to the wire beneath thechannel generates heat under the edge of the droplet. Heating the leftinterface increases the internal pressure on that end of the droplet andforces the entire droplet to the right. The pressure on the interior ofthe droplet may be calculated knowing the atmospheric pressure, P_(atm),surface tension, σ, and the dimensions of the channel. For a circularcross-section, the interior pressure, P_(i), is given by P_(i)=P_(atm)(4σ cos θ)/d where d is the diameter of the channel and θ is the contactangle. Since σ is a function of temperature (σ=σ_(o)(1−bT) where σ_(o)and b are positive constants and T is the temperature), increasing thetemperature on the left end of the droplet decreases the surface tensionand, therefore, increases the internal pressure on that end. Thepressure difference between the two ends then pushes the droplet towardsthe direction of lower pressure (i.e., towards the right). The aqueousdroplet shown is in a hydrophilic channel (0<θ<90); for a hydrophobicchannel (90<θ<180), heating the right edge would make the droplet moveto the right.

[0234] Contact angle hysteresis (the contact angle on the advancing edgeof the droplet is larger than the contact angle on the retreating edge)requires a minimum temperature difference before movement will occur.The velocity of the droplet after motion begins may be approximatedusing the equation ν=AEPd²/32 μL where AEP is the pressure difference, μis the viscosity of the solution, and L is the length of the droplet.The present invention contemplates temperature differences of greaterthan 30° C. to create movement. Studies using temperature sensorsarrayed along the entire channel indicate that a differential ofapproximately 40° C. across the droplet is sufficient to provide motion.In these studies, the channel cross-section was 20/μm×500/μm, and thevolume of each of these droplets may be calculated from their lengthsand is approximately 100 nanoliters for a 1 cm long droplet.

[0235] B. Integrated Fluid Handling System

[0236] Although there are many designs currently available for liquidhandling in micromachined devices, a preferred method uses individualdrops propelled by induced gradients in surface tension. The principlebehind the technique is to inject the samples into the device asdiscrete drops. These drops, once inside the channels, may be propelledby changing the forces on the two drop surfaces. For instance, if thedrops are in a hydrophilic channel (glass), the interfacial tension actsoutward from both ends. Since the surface tension of water decreaseswith increasing temperature, heating the left side of the drop causesthe drop to be propelled towards the right. Splitting, merging, andmixing of such drops may also be accomplished by careful control of droplocation in micromachined channels.

[0237] In certain embodinments, the channels contain approximately 30heaters and 10 temperature sensors along the length of the channels.Location sensors can sense the location, length, and, therefore, thevolume of the drop. The base material of the chip is silicon withsilicon oxide and nitride layers used for insulation. The resistiveheaters in the channel may be made from a variety of materials includingplatinum, aluminum, and doped polysilicon; in one aspect the chip hasgold heaters. These resistive heaters are inlaid into the insulatingoxide to provide a smooth (<1 μm) surface for the upper insulatinglayer: failure to make the upper surface smooth can result in layerinstabilities and device failure during heating in an aqueousenvironment. Silicon or glass channels may be attached to the substratewith a variety of adhesive techniques. Anodic, UV cure cement, andpolyimide bonding have been used in the invention, though other methodsmay be used, and are known to those of skill in the art.

[0238] Drop motion was induced by changes in the surface tension in aglass channel glued to a silicon substrate using UV-cure cement. Notethat the surface conditions, solution conditions, and channel geometry,all affect the motion of the drop. Careful attention must be paid toboth the construction procedure and the surface preparation procedure ordrop motion will not occur. By being able to move drops in this manner,the mixing of two drops (for sample injection) or the splitting of onedrop into two (for post reaction treatment) may be accomplished.

[0239] C. Characteristics of Micro-Scale Fluids

[0240] In miniaturization of a DNA processing system, most componentsmay be designed similar to their benchtop equivalents. Fabrication atthe micron level may then be accomplished using known siliconcharacteristics. Some specific components are easily miniaturized. Forinstance, a heating element on a silicon wafer will, for mostapplications, be able to heat a sample much more rapidly than a largerscale heater. This increase in efficiency is due to a decrease in thedistance over which thermal energy must travel and to the reduced massof the sample being heated.

[0241] In contrast to the heating of samples, the movement of samples ismore complicated. The diameter of the “tubing” through which sampleswill flow in the proposed system may be reduced to a channel width assmall as 10 μm. This extremely small diameter will change the typicalcharacteristics of the fluid flow. Methods to move liquids become muchmore difficult in the nanoliter volume range. The Reynold's number (Re)of a liquid system is an indication of the ease with which a liquid willmove, and is defined as Equation 1:

Re=(ν)(d)(r/μ)  (1)

[0242] where ν is the velocity, d is the diameter of the tubing, r isthe density of the solution, and μ is its viscosity. Using the Reynold'snumber, comparing a 1 cm diameter tube to a 10 μm diameter channel wouldresult in a Re decrease from about 100 to 10⁻² (for water moving with avelocity of 1 cm/s).

[0243] One method of coping with this new flow regime (very low Re)would be to use higher pressure pumps. High pressure liquidchromatography (HPLC) system (d˜10 μm) typically run at thousands ofpounds per square inch (PSI) pressure, while standard liquidchromatography systems (d˜100 μm) can operate with only several PSI. Inthe silicon wafer system, a pump-based propulsion mechanism may befabricated by designing an “in-chip” peristaltic pump (Folta et al.,1992). This micropump design consists of a heating element within athermopneumatic chamber. The thernopneumatic chamber, when heated,causes a membrane along the flow channel to “bulge”. Peristaltic pumpingoccurs by “bulging” of a set of thermopneumatic actuators in series (VanLintel, 1988; Pohl, 1978). Unfortunately, these pumps must generate arelatively high pressure to move the liquids through micrometer-sizedtubing.

[0244] Another method for moving small volumes of liquid is to usegradients in surface tension (Edwards et al., 1991). If a thincapillary, tube is inserted into water, the liquid in the capillary,will rise a centimeter or so above the surface of the surrounding water.This rise is due to the force of surface tension acting on the meniscusas defined by Equation 2:

F=(π)(d)(σ)(cos θ)  (2)

[0245] where d is the inside diameter of the tube, σ is the surfacetension (force/length) and θ is the contact angle (Osipow, 1962). If asmaller diameter capillary is used, the decrease in force isproportional to d but the decrease in weight of water per unit height inthe capillary decreases by d². Therefore, for very large diameter tubes,the forces of surface tension can usually be neglected due to the largemass of fluid. However, for small tubes, pores, or channels, the forceof surface tension may be great compared to the mass of liquid beingmoved. This “wicking” of liquid is a common occurrence and may beobserved when a porous solid is brought in contact with a solution(i.e., paper towel and water).

[0246] By controlling the magnitude and direction of the surface forces,the movement of small sample volumes in the interior of capillary tubesmay be controlled. Several researchers have described moving small dropsthrough silicon channels using this principle (Beni and Tenan, 1981;Matsumoto and Colgate, 1990; Fuhr et al., 1992). The technique may bebest understood by examining the liquid drop contained in a glasscapillary. For glass, θ=0, consequently, the force due to surfacetension is pulling the drop both to the right and to the left, and isperfectly balanced. If the surface tension on the left side of the dropis decreased or the surface tension on the left side is increased, thedrop will be pulled to the right. Movement to the left may beaccomplished in a similar fashion (decreasing the surface tension on theleft or increasing the surface tension on the right). Most previous workhas changed the surface tension force by altering the channel wallhydrophobicity, and consequently, the contact angle θ.

[0247] Alternatively, the surface force may be altered through changesin the liquid surface. It is known that the surface tension of liquidsis a strong function of both temperature and surface electrical charge(Osipow, 1962). Matsumoto and colleagues used electrostatic control todevelop a surface tension driven micropump (Beni and Tenan, 1981;Matsumoto and Colgate, 1990). Because of the possibility of chargeattraction with the DNA molecules in solution, temperature control maybe a preferred choice for changing liquid surface tension in theinvention. For most liquids, surface tension decreases nearly linearlywith increased temperature.

[0248] Modeling this dependence may be accomplished using the linearempirical expression. Equation 3:

σ=σ_(o)(1−bT)  (3)

[0249] where σ_(o) and b are constants (Beni and Tenan, 1981; Matsumotoand Colgate, 1990). This expression means that increases in temperatureresult in linear decreases in surface tension. An empirical model wasobtained by a linear fit of σ_(o) versus temperature for pure water. Thechange in surface tension with temperature for pure water isapproximately −0.16 dyne/cm (Probstein, 1989) and remains nearlyconstant over all temperatures for liquid water. It is the magnitude ofthis change that can serve as the driving force for fluid movement;therefore, knowledge of this parameter's magnitude is necessary forpredictions of liquid velocities in a capillary system.

[0250] D. Micro-Scale Fluid/Solute Parameters

[0251] As demonstrated in Equation 4, the velocity profile for fluidmotion in a capillary tube depends upon several characteristics of theliquid and its interface with the flow chamber. These include: Δσ, thesurface tension difference between the drop ends, d, the capillarydiameter, μ, the liquid viscosity, and L the drop length.

ν_(ave)(Δσ)(d)/(8)(μ)(L)  (4)

[0252] For example, the liquid viscosity, the surface tension differencebetween the ends of the drop, and the contact angle between the liquidand the flow chamber all influence the magnitude of fluid motion.However, the flow chamber dimensions and geometry affect the shape ofthe velocity profile of the liquid.

[0253] E. Fluid Viscosity

[0254] Fluid rheology is the study of how a fluid reacts to a stress(force/area). For instance, a common class of fluids, termed Newtonianfluids, exhibit a regular response to a fluid stress. The behavior of aNewtonian fluid may be expressed in terms of its constitutive equation,which states that the shear stress (force/area) is proportional to thelocal velocity gradient (Bird et al., 1960):

Shear Stress=(viscosity)(velocity gradient)  (5)

[0255] where the constant of proportionality, the viscosity of thefluid, is an indication of the resistance to flow.

[0256] The dilute aqueous DNA solutions are stored in a Tris-HCl (10mM), EDTA (1 mM) buffer solution. Although the DNA concentration ispresumably too low to affect the physical behavior of the macroscopicfluid, the Newtonian behavior of the water-based solvent was checked.Viscosity measurements, as a function of shear rate, were taken for pureTris-HCl/EDTA buffer in addition to 1, 50, and 105 microgram/ml samplesof DNA solution. The results indicate Newtonian behavior for all threeDNA concentrations and for the Tris-HCl/EDTA buffer. As expected, theviscosities of four samples tested were Newtonian, and had viscositiesvery close to that for water at 25° C. (ie., lcP).

[0257] F. Surface Characteristics

[0258] The contact angle (force/contact length) between a fluid and itssolid surface is an extremely important parameter in surface tensiondriven flow. The magnitude of this force is directly related to thecosine of the contact angle between the liquid and the solid flowchamber. To maximize this force, a perfectly hydrophilic (contact angleof 0°, cos 0°=13 or perfectly hydrophobic (contact angle of 180°, cos180°=−1) surface is preferred. For example, clean glass surfaces areextremely hydrophilic and form a 0° contact angle with pure waterproducing the maximum surface tension. Surface treatments of glass canproduce hydrophobic surfaces. Two hydrophobic glass surface treatmentprocesses have been examined. First, a silane treatment was followed byaddition of a long chain aldehyde (decyl aldehyde). In the secondtreatment, a commercial brand Rain-X used. Interestingly, the Rain-Xtreatment was the easiest to apply and produced a more hydrophobicsurface than the silane treatment. However, the Rain-X contact angle wasstill much less than optimal 180° making it a less than ideal surfacefor surface tension driven flow.

[0259] G. Surface Tension

[0260] From equation 4, the change in surface tension can serve as thedriving force for fluid motion. One embodiment of the invention isdescribed as a micromechanical integrated DNA analysis technology, orMIDAT. In the MIDAT system a temperature difference between the ends ofthe drop will be used to produce a surface tension difference. For purewater, the change in surface tension with temperature is −0.15 dyn/cm−°C. (Probstein, 1989) and is constant over the entire liquid range ofwater (Osipow, 1962). Because the DNA solutions being used are verydilute, the surface tension values are expected to be identical towater.

[0261] Using the Krus Interfacial Tensiometer K8, the surface tension ofboth pure water and buffer solution was measured at several temperaturesbetween 15° C. and 55° C. As expected, the buffer and the watersolutions exhibit nearly identical slopes. Also, the surface tensions ofDNA solutions at several concentrations between 0 and 120 ug/ml weremeasured. The DNA concentrations were chosen to reflect a range relevantto standard PCR™ reaction conditions (1 μg/ml, 50 μg/ml and 105 μg/ml).There is little to no change in surface tension with varying DNAconcentrations at 25° C. These values, ranging from 70-71 dyn/cm, arevery close to those described for pure water at 25° C. (72 dyn/cm).

[0262] H. Capillary Drop Movement

[0263] As an initial demonstration of surface tension driven flow, asmall volume of water was moved in a 0.5 mm inside diameter glasscapillary. This was accomplished by heating one of the liquid to airinterfaces on the drop, thereby imbalancing the surface tension presentin the two surfaces of the drop. The 1.5 centimeter long drop was movedapproximately 3 cm forward and back using a hot water spray (80° C.) asa heating system. The spray was washed over the glass capillary near theback end of the drop, and followed the drop as it moved. A roughestimate of the velocity of the drop may be calculated from the timedvideo images. The drop is moving at approximately 0.5 cm/sec, which isof the same order of magnitude as the theoretical velocity prediction,as calculated from Equation 4.

[0264] I. Silicon Microfabrication and Integrated Systems.

[0265]FIG. 2B shows a physical layout of a constructed chip. It consistsof a two wafer bonded structure. One of the wafers is made of siliconand the other is glass. In the glass wafer, two levels of thin-filmaluminum are patterned to make electrodes, interconnects, and heatersfor the driven mechanisms. On the silicon wafer, the chip is patternedwith microchannels and sample inlets and outlets. The two wafers arebonded together to complete the system. The sample is moved inside thechannel using a linear array of electrical devices.

[0266] Three propulsion mechanisms are contemplated other than thethermal surface tension method for fluid propulsion chips. Microchannelswith electrowetting (Beni and Tenan, 1981; Matsumoto and Colgate, 1990;Washizu, 1992), dielectrophoretic, and thermal gradient (Van Lintel,1988; Pohl, 1978) drives have been fabricated. Briefly, electrowettingpropulsion relies on charge-induced change in the hydrophobicity(wetting) characteristics of the channel wall. Induction of a currentalong the channel makes the wall more hydrophilic, drawing the liquiddrop toward the activated electrode. Dielectrophoresis utilizes thedifference in dielectric constant between water and air. A liquid dropwill be preferentially draw in between the plates of a chargedcapacitor. Each chip is designed to move samples in the 5-50 nL range.

[0267] Twenty-seven devices were fabricated from a single 100 mmdiameter wafer. The chips are cut out of the wafer and bonded to aprinted circuit (PC) board. The setup is constructed to control thesignals to each microchannel heater or electrode using a computer forsample droplet formation, separation, and movement control. The completefabrication process requires 5 lithographic steps and was completed inone week. Microchannels with thicknesses of 20, 50, 100, and 200 μm deepand 500-1000 μm width were patterned. Each of these was fabricated with20, 50, or 150 electrodes along the microchannel length.

[0268] In determining whether a drop will move due to surface tensiongradients, the two key parameters are the magnitude of the surfacetension (σ) and the contact angle (θ). For channels in silicon wafers,the surface is easily oxidized, producing a glass-like surface whosecontact angle is approximately zero. This implies that the surface ishydrophilic and the liquid will “wet” the walls of the channel. For adrop in this channel, a force balance on a horizontally oriented dropgives

(π)(d)(σ_(left))=(π)(d)(σ)_(right)  (6)

[0269] from equation 2. Since the surface tension is constant for aliquid at constant temperature, the forces on each side of the drop areidentical, thus the drop remains motionless.

[0270] Knowing that surface tension is a function of temperature, thesurface tension on one side of the drop may be selectively changed. Thesurface tension of water decreases as the temperature at theliquid-solid interface increases. Therefore, using a microheater locatedslightly beneath the surface of the channel, the surface tension on oneside of the drop may be reduced while keeping the other side constant.Using the heater in combination with a thermocouple (or otherthermosensor), the temperature, and therefore the surface tension, atthat edge may be accurately controlled. The unequal heating willaccelerate the drop away from the heat source. Sensors fabricatedbeneath the channel may be used to locate the edge of the drop.(Dielectric sensors may be used for this application, as the dielectricconstant of water is different from that of air.) By sensing thismovement and turning on sequential heating elements at the rear edge ofthe moving drop, the drop may be propelled down the flow channel in a“bucket brigade” fashion. Sequence control of the heater activation maybe performed by quadrature electrical signals.

[0271] The velocity at which the drop will move may be determined bybalancing the force generated by the surface tension gradient with thedrag caused by the fluid flowing through the channel. The averagesteady-state velocity for pressure-driven flow in a capillary tube,termed Poiseuille flow, is given as equation 7:

ν_(ave)=[(ΔP)(d ²)]/[(32)(μ)(L)]  (7)

[0272] where ΔP is the pressure difference between the drop ends, d isthe capillary diameter, μ is the liquid viscosity and L is the droplength (Bird et al., 1960). The pressure difference is a result of thecurvature at the interface. Use of Young-Laplace equation relating thepressure difference across a curved interface (Probstein, 1989), such asin a hydrophilic capillary system, results in Equation 4 for calculatingthe average steady-state velocity for surface tension driven flow:

ν_(ave)=[(Δσ)(d)]/[(8)(μ)(L)]  (4)

[0273] where Δσ is the difference in surface tension between the ends ofthe drop. Thus for only small temperature differences across the drop(on the order of 10° C.) velocities on the order of 1 cm/s may beobtained. This velocity is more than sufficient for transporting liquiddrops in MIDAT and other chip based systems.

[0274] It should be noted that other methods also exist for moving adrop by changes in surface tension. The drop may be moved by changingthe hydrophobicity of the channel surface (electrowetting). The surfacemay be made hydrophobic by a variety of chemical surface treatments.Imparting an electrostatic charge to the channel wall surface at theright edge of the drop, and thereby decreasing the contact angle willhave the same effect as heating it: the drop will move to the right.These methods are particularly attractive as they are not greatlyeffected by the low Reynold's numbers associated with moving smallliquid volumes.

[0275] J. MIDAT Channel Injection

[0276] Accurate and reproducible injection of a small liquid volume intomicromachined channels may be accomplished using these principles. Atthe sample injection port, the channel immediately adjacent to the inputreservoir contains a section that is hydrophobic. This portion of thechannel contains a series of electrodes that can change the channelhydrophobicity (electrowetting).

[0277] A drop of solution is placed on the hydrophilic input reservoirto form a sessile drop. The reservoir is connected to the entrance of amicrochannel that delivers the sample into the MIDAT device. A portionof the channel is then made hydrophilic by charging a set of electrodes.Once the required amount of liquid is drawn into the device, the regionnear the junction of the reservoir and the channel is made locallyhydrophobic by turning off the most proximal electrode. Alternatively, abrief burst of heating at the junction could vaporize a small quantityof the sample and break the continuity of the drop. In eitherconfiguration, no further in-flow of liquid occurs. The drop is thenmoved forward by electrowetting or thermal surface tension effects, asdiscussed previously. Replacement air is drawn in through a smallhydrophobic-coated channel. The volume of the drop is fixed by thecross-sectional area of the channel and the distance between cleavagepoint and the leading drop edge. Sample volumes as low as 10⁻¹² litersmay be manipulated by this system.

[0278] During the movement of solutions, the biological activity of thesamples must be preserved. Since the channel may be designed to almostany dimension, the surface area/volume ratio may be kept low to avoidsurface denaturation of reaction mixture protein components (i.e., DNApolymerase). Adsorption of solutes onto the surface of the channels mustbe minimized and may be controlled by proper treatment of the channelwith various dopants. Conversely, the desorption of silicon dopants intothe reaction solutions must be carefully monitored, as these may affectbiochemical reactions.

[0279] When it is necessary to move the fluid within the channels orchambers of the device, pressure (e.g., air pressure) may be applied toan opening in the channel or chamber (e.g., the inlet port). Whenpressure is used to move the liquid, there is preferably a secondopening or exit port which may be used to apply pressure in the oppositedirection or to remove the liquid from the device. Alternatively, thefluid may be moved within the channel using a thermocapillary pump asdescribed by Burns, et al. (1996). The thermocapillary pump has theadvantage of providing a self-contained miniaturized device in whichmovement of discrete aliquots within the channels requires no movingparts or valves.

[0280] IV. Flow Control with Sealed Valves

[0281] The present invention contemplates the use of sealed valves tocontrol fluid flow. While the present invention is not limited to aparticular sealing method, in one embodiment, an actuating force pushesa diaphragm against a valve seat to restrict fluid flow and thediaphragm is then sealed to the valve seat. In such an embodiment, thesolder pads are associated with a heating element that can melt thesolder. This liquefied solder flows over areas of the valve seat anddiaphragm to cover contamination, cracks and crooks between thediaphragm and valve seat. With the actuating force still holding thediaphragm and valve-seat together, the heating element is turned off toallow the solder to cool and re-solidify. Upon solidification, theactuating force may be released and the valve is sealed. To open thevalve again, the solder may be liquefied without applying an actuationforce.

[0282] In certain aspects of the invention “diaphragm” may refer to anelement capable of being manipulated such that it can at least partiallyblock the passage of fluid in a channel in one position (extended) andpermit the flow of fluid in a channel in another position. An “actuatingforce” is a force that is capable of extending a diaphragm. A “valveseat” is an element designed to accept a portion of the diaphragm whenextended. A “movement means” is a means capable of moving liquefiedmeltable material (e.g., force air, magnetic field, etc.).

[0283] In a preferred embodiment, the valve is designed such that solderpads are placed on the diaphragm or valve seat. While the presentinvention is not limited to a precise method of placing these solderpads, it is specifically contemplated that they may be electroplated.

[0284] V. Mixing Biological Samples in Reactions

[0285] Droplet motion (described generally above) is contemplated as onestep in a pathway. The other steps typically involve sample mixing and acontrolled reaction. For example, the integral heaters arrayed along theentire surface of the channel used for droplet motion also allow for aregion of a channel to be used as a thermal reaction chamber. For samplemixing prior to the reaction, a Y-channel device is one embodiment ofthe invention. In such a device, a first droplet containing a firstsample (e.g., nucleic acid) is moved along one channel of the Y-channeldevice, and a second droplet containing a second sample (e.g., arestriction digest enzyme in digestion buffer) is moved along the otherchannel of the Y-channel device.

[0286] Following sample merging there is the concern that the combinedsamples have not been properly mixed. That is to say, if two similarmicrodroplets enter the single channel in laminar flow at the same flowrate, they will form an axially uniform droplet but will not be mixedwidth-wise. Width-mixing may be accomplished in a number of ways.

[0287] First, there is diffusion, although, for large DNA molecules, thecharacteristic time for this mixing could be on the order of severalhours or more. Circulation patterns generated inside the droplets duringmovement and heating significantly reduce this time. In this regard, thepresent invention contemplates maintaining the mixture as a heatedmixture (e.g., maintaining the temperature at 65° C. for 10 min) usingthe integral heaters and temperature sensors.

[0288] Second, the present invention contemplates mixing by reversingthe flow direction of the mixture over a relatively short distance inthe channel. While a variety of reverse flow approaches are possible,one or two direction changes over a distance comprising approximatelytwo droplet lengths has been found to be adequate.

[0289] Finally, there is the mixing approach wherein the mixture ismoved against or over physical obstacles. For example, the mixture maybe either “crashed” back against merge point of the Y-channel or simplymoved over deliberate imperfections in the channel (i.e., “rollercoaster” mixing).

[0290] Successful mixing, of course, may be confirmed bycharacterization of the product(s) from the reaction. Where product isdetected, mixing has been at least partially successful. The presentinvention contemplates, in one embodiment, using electrophoresis toconfirm product formation.

[0291] A restriction digest was performed by mixing a DNA sample with anenzyme solution and heating the resulting mixture. The solutions wereinjected into the ends of the y-channel (simple capillary action drewthe samples into the channels). The drops were then moved using theembedded heaters and, once the combined drop was in the single channel,it was heated to a constant temperature. Comparison of the results ofthis digestion with that performed on commercial thermocyclers indicatedlittle difference.

[0292] A. Biocompatibility

[0293] A 5 mm×5 mm heater surface for is used as a polymerase chainreaction thermocycler. The cross-shaped loop that divides the regioninto four heating zones is an RTD (resistive temperature detector). Theconstruction of this chamber is identical to the y-channel heatersdescribed earlier. In fact, the chips are processed on the same wafers.Reactions may be carried out on this surface using either walled polymervessels for large-volume reactions or etched caps for small-volume (˜0.5μl) reactions.

[0294] PCR™ was run on this chip. The reaction was carried out on thesurface of this chip using a polypropylene ring cemented to the chip asthe vessel walls. 20 gm of reaction mix was covered with oil to preventevaporation and the solution was cycled through 94° C., 55° C., and 72°C. using a digital controller (National Instruments LabView, programmedVI, Macintosh Quadra 950 computer). Using such a controller based inLabView allows change in the function and design of the controllerwithout the expense of circuit construction. As the electrophoresis gelindicates, the oxide surface of the chip and the heaters did not damagethe enzyme or inhibit the reaction; the chip results appear identical tothe control run on a commercial thermocycler. Extensive biocompatibilitytests indicate that the results of the reaction are very sensitive tocontroller settings and to the materials used for construction (Burns,1994).

[0295] B. Reaction Parameters

[0296] Solutions containing the DNA samples and solutions containing thereagents for the reaction must both be added to the MIDAT unit,thoroughly mixed, and reacted at the proper temperature. The mixing ofsolutes at very small length scales is both simple and complex. Thesimplicity arises because the radial distance that the solutes mustdiffuse is relatively small and, therefore, any radial mixing will occurquite rapidly. For instance, in a 1 μm channel, the characteristic timefor diffusion a typical solute (D=10⁻⁵ cm²/s) is approximately(Probstein, 1989):

t=L ² /D=(10⁻⁴ cm)²/(10⁻⁵ cm ² /s)≈1 ms  (8)

[0297] Even for larger solutes with diffusion coefficients of 10⁻⁶ to10⁻⁸ cm²/s, the mixing time is under 1 s.

[0298] The complexity arises because the mixing lengthwise in a channelof length 1 cm or longer is not rapid (t≈several days). Care must betaken to assure that uniform mixing occurs along the length of a drop ofsolution. This uniform concentration may be assured in several ways.First as two drops are mixed, they will join at the front ends. Thisjoining may be accurately controlled using dielectric sensors andheaters, as discussed before. If the drops are in a hydrophilic channel,each meniscus will naturally join. By controlling the force driving eachdrop, the liquids may be added at precisely the rate to yield a uniformaxial concentration.

[0299] In addition to this precise control of the liquid motion, uniformaxial concentrations may be assured due to the flow pattern generatedwithin the drop moving in the channel. The liquid near the surface ofthe channel, due to intermolecular forces, remains motionless whileliquid in the center of the drop is moving forward at twice the averagevelocity of the drop. At the back edge of the drop, this stagnant liquidis “picked off” the walls of the channel by surface tension while at thefront of the drop, the liquid is deposited by surface wetting phenomena.In this way, the liquid is constantly circulating from the front of thedrop, down the side of the drop, and returning through the center.During this travel, the solutes are rapidly mixing radially with thedifferent velocity streams. The net result is that, as long as the droptravels approximately one or two drop lengths, complete mixing of thedrop should occur.

[0300] VI. Isothermal Amplification Reactions

[0301] A. Enzymatic Reactions

[0302] The channels of the DNA chip may be constructed in anyconfiguration appropriate for the selected reaction protocol. Thecomplete amplification reaction, including the target and the othercomponents for the amplification reaction, may prepared and mixedoutside of the DNA chip. The complete amplification reaction is thenplaced into the channel of the DNA chip and, if necessary, moved to aregion of the channel in contact with a heater element which maintainsthe desired reaction temperature. Alternatively, the reaction may beperformed in a device in which the sample containing the target and thesample containing the enzymes and other components of the amplificationreaction are maintained as separate liquid aliquots until the reactionis to be initiated. At that time, the two liquid aliquots may be broughtinto contact by means of pressure, a thermocapillary pump, otherequivalent means, such that they mix and react in a region of thechannel which is maintained at the desired reaction temperature by aheater element. In an alternative embodiment, the channels of the DNAchip may be in the form of a “Y” such that a liquid aliquot containingthe target placed in one arm of the “Y” is kept separate from theenzymes and amplification reagents in the other arm of the “Y”. Usingpressure applied to the inlet port of each arm of the “Y” orthermocapillary pumps in contact with each arm, the two liquid aliquotsare moved into contact at the junction of the two arms and allowed tomix and react at a selected reaction temperature maintained by theheater element in the region of the channel which forms the stem of the“Y”. Other configurations for the channels and device designs which alsoemploy reaction chambers and/or detection areas will be apparent tothose skilled in the art and are intended to be included within thescope of the invention. If desired, mixing may be enhanced by moving theliquid aliquot back and forth by alternately applying pressure on eachside or alternately heating each side of the aliquot, though otherequivalent means may be substituted.

[0303] In certain preferred embodiments of the invention, the device foruse in the isothermal amplification of a selected nucleic acid furthercomprises one or more of the reagents for an isothermal nucleic acidamplification reaction. Such reagents may include polymerases,nucleotides, buffers, solvents, nucleases, endonucleases, primers,target nucleic acids including DNA and/or RNA, salts, and other suitablechemical or biological components. In certain preferred aspects, thesereagents may be provided in dry or lyophilized form. In otherembodiments the reagents may be dissolved in a suitable solvent.

[0304] In certain embodiments one or more of the reagents, includingnucleotides, buffers, salts, chemicals, solvents, primers, targetnucleic acids including DNA and/or RNA, polymerases, endonucleases,nucleases, and chemical or biological components suitable for theisothermal reaction mixture are added to the at least first and/orsecond microdroplet transport channels separately or in variouscombinations. In other preferred embodiments of the invention one ormore of the nucleotides, buffers, salts, chemicals, solvents, primers,target nucleic acids including DNA and/or RNA, polymerases,endonucleases, nucleases, and chemical or biological components suitablefor an isothermal amplification reaction are contained in, in liquidcommunication with, operably or functionally connected to, and/orprovided with the microfabricated substrate. In certain otherembodiments one or more or the reagents for an isothermal reaction maybe contained in a detachable reservoir that may be contained in orattached to an inlet port, channel, or reservoir so to be in liquidcommunication with, and/or operably or functionally connected to themicrofabricated substrate. In certain preferred embodiments the reagentsmay be in dry or lyophilized form. In other embodiments the reagents maybe dissolved in a suitable solvent.

[0305] Any isothermal nucleic acid amplification method may be performedon the DNA chips essentially as described in the art. The lower,constant temperature and complex enzymology of isothermal amplificationdoes not inhibit the reaction in the DNA chip format. That is, it hasunexpectedly been found that movement and mixing of the liquid aliquotsis not significantly compromised, that the enzymes involved inisothermal amplification are not significantly inhibited, and that thepredicted stagnant temperature gradient does not prevent efficientamplification. Thermophilic SDA (tSDA) is a preferred amplificationmethod for application to DNA chips because of its high amplificationfactors and rapid results. Amplification reactions are generallyperformed in the microfabricated device in a volume of about 0.6 μL-3μL, but the dimensions of the channels and/or reaction chambers may bealtered to accommodate larger or smaller reaction volumes.

[0306] Nucleic acid used as a template for amplification is isolatedfrom cells contained in the biological sample, according to standardmethodologies (Sambrook et al., 1989). The nucleic acid may be genomicDNA or fractionated or whole cell RNA. Where RNA is used, it may bedesired to convert the RNA to a complementary DNA. In one embodiment,the RNA is whole cell RNA and is used directly as the template foramplification.

[0307] Pairs of primers that selectively hybridize to nucleic acidscorresponding to an isolated target sequence for amplification arecontacted with the isolated nucleic acid under conditions that permitselective hybridization. The term “primer”, as defined herein, is meantto encompass any nucleic acid that is capable of priming the synthesisof a nascent nucleic acid in a template-dependent process. Typically,primers are oligonucleotides from ten to twenty base pairs in length,but longer sequences can be employed. Primers may be provided indouble-stranded or single-stranded form, although the single-strandedform is preferred.

[0308] Once hybridized, the nucleic acid:primer complex is contactedwith one or more enzymes that facilitate template-dependent nucleic acidsynthesis. Multiple rounds of amplification, also referred to as“cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

[0309] Next, the amplification product is detected. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electrical orthermal impulse signals (Affymax technology).

[0310] B. Types of Nucleic Acid Amplification

[0311] A number of template dependent processes are available to amplifynucleotide sequences present in a given template sample. It is notintended that the present invention be limited by the nature of thereactions carried out in the microscale device. Reactions include, butare not limited to, chemical and biological reactions. Biologicalreactions include, but are not limited to sequencing, restriction enzymedigests, RFLP, nucleic acid amplification, and gel electrophoresis. Itis also not intended that the invention be limited by the particularpurpose for carrying out the biological reactions. In one medicaldiagnostic application, it may be desirable to differentiate between aheterozygotic and homozygotic target and, in the latter case, specifyingwhich homozygote is present. Where a given genetic locus might code forallele A or allele a, the assay allows for the differentiation of an AAfrom an Aa from an aa pair of alleles. In another medical diagnosticapplication, it may be desirable to simply detect the presence orabsence of specific allelic variants of pathogens in a clinical sample.For example, different species or subspecies of bacteria may havedifferent susceptibilities to antibiotics; rapid identification of thespecific species or subspecies present aids diagnosis and allowsinitiation of appropriate treatment.

[0312] Preferred methods are Strand Displacement Amplification (SDA) andthermophilic SDA for carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation.

[0313] In this method, either before or after the template nucleic acidsare denatured, a mixture comprising an excess of all fourdeoxynucleosidetriphosphates, wherein at least one of which issubstituted, a polymerase and an endonuclease are added. (If hightemperature is used to denature the nucleic acids, unless thermophilicenzymes are used, it is preferable to add the enzymes afterdenaturation.) The substituted deoxynucleosidetriphosphate should bemodified such that it will inhibit cleavage in the strand containing thesubstituted deoxynucleotides but will not inhibit cleavage on the otherstrand. Examples of such substituted deoxynucleosidetriphosphatesinclude 2′deoxyadenosine 5′-O-(1-thiotriphosphate),5-methyldeoxycytidine 5′-triphosphate, 2′-deoxyuridine 5′-triphosphateand 7-deaza-2′-deoxyguanosine 5′-triphosphate.

[0314] The mixture comprising the reaction components for targetgeneration and SDA can optionally include NMP (1-methyl 2pyrrolidinone), glycerol, polyp(ethylene glycol), dimethyl sulfoxideand/or formamide. The inclusion of such organic solvents is believed tohelp alleviate background hybridization reactions.

[0315] It should be appreciated that the substitution of thedeoxynucleotides may be accomplished after incorporation into a strand.For example, a methylase, such as M. Taq I, could be used to add methylgroups to the synthesized strand. The methyl groups when added to thenucleotides are thus substituted and will function in similar manner tothe thiosubstituted nucleotides.

[0316] It further should be appreciated that if all the nucleotides aresubstituted, then the polymerase need not lack the 5′ forward arrow 3′exonuclease activity. The presence of the substituents throughout thesynthesized strand will function to prevent such activity withoutinactivating the system.

[0317] The selection of the endonuclease used in this method should besuch that it will cleave a strand at or 3′ (or 5′) to the recognitionsequence. The endonuclease further should be selected so as not tocleave the complementary recognition sequence that will be generated inthe target strand by the presence of the polymerase, and further shouldbe selected so as to dissociate from the nicked recognition sequence ata reasonable rate. It need not be thermophilic. Endonucleases, such asHincII, HindII, AvaI, Fnu4HI, Tth111I, and NciI are preferred.

[0318] One can envision several alternative nicking enzyme systems inaddition to those detailed in this application. For example, it isgenerally regarded that class IIS restriction endonucleases (e.g., Fokl)contain two DNA cleavage centers within a single polypeptide unit. Ifone of the cleavage centers was inactivated, such as through sitedirected mutagenesis, the resultant nicking enzyme could be used in anamplification system not requiring modifieddeoxynucleosidetriphosphates. As an additional example, the restrictionenzyme EcoRi has been shown to preferentially cleave one strand innoncanonical recognition sites or when its canonical recognition site isflanked by an oligopurine tract (Thielking et al., 1990; Lesser et al.,1990; Venditti & Wells, 1991). As another example, the restrictionenzyme DpnI (available from New England Biolabs, Beverly Mass.) cleavesa recognition site containing me⁶ dA on both strands. DpnI or ananalogous restriction enzyme may be able to nick the methyl containingstrand of a hemimethylated recognition site. Such a system would employSDA primers (P₁ and P₂) with methylated recognition sequences along withunmodified deoxynucleosidetriphosphates. Alternatively, certainrestriction enzymes are known to cleave the nonmethylated strand of ahemimethylated recognition site (e.g., MspI and me⁵ dC). Such a systemwould use a methylated deoxynucleosidetriphosphate. Finally, one coulduse origin of replication proteins to nick one strand of a recognitionsequence.

[0319] Polymerases useful in this method include those that willinitiate 5′-3′ polymerization at a nick site. The polymerase should alsodisplace the polymerized strand downstream from the nick, and,importantly, should also lack any 5′ forward arrow 3′ exonucleaseactivity. It should be appreciated that a polymerase ordinarily havingsuch exonuclease activity may be deemed to “lack” such activity if thatactivity is blocked by the addition of a blocking agent.

[0320] An additional feature of this method is that it does not requiretemperature cycling. Many amplification methods require temperaturecycling in order to dissociate the target from the synthesized strand.In this method, a single temperature may be employed after denaturationhas occurred. The temperature of the reaction should be high enough toset a level of stringency that minimizes non-specific binding but lowenough to allow specific hybridization to the target strand. In additionproper temperature should support efficient enzyme activity. From about37° C. to about 42° C. has been found to be a preferred temperaturerange.

[0321] The SDA reaction initially developed was conducted at a constanttemperature between about 37° C. and 42° C. (U.S. Pat. No. 5,455,166,incorporated herein by reference,). This was because the exo⁻ Klenow DNApolymerase and the restriction endo nuclease (e.g., Hindu) aremesophilic enzymes which are thermolabile (temperature sensitive) attemperatures above this range. The enzymes which drive the amplificationare therefore inactivated as the reaction temperature is increased.

[0322] Methods for isothermal Strand Displacement Amplification whichmay be performed in a higher temperature range than conventional SDA(about 50° C. to 70° C., “thermophilic SDA”) were later developed.Thermophilic SDA is described in published European Patent ApplicationNo. 0 684 315 and employs thermophilic restriction endonucleases whichnick the hemimodified restriction endonuclease recognition/cleavage siteat high temperature and thermophilic polymerases which extend from thenick and displacing the downstream strand in the same temperature range.At increased temperature, the amplification reaction has improvedspecificity and efficiency, reduced nonspecific backgroundamplification, and potentially improved yields of amplificationproducts. In addition, the need to add the enzymes in a separate stepafter the initial heat denaturation of double stranded targets may beeliminated when enzymes which are stable at the denaturation temperatureare used. UDG decontamination of target-specific amplicons in the SDAreaction is also more efficient when the amount of nonspecificbackground amplicons is reduced.

[0323] Another method, called Repair Chain Reaction (RCR), involvesannealing several probes throughout a region targeted for amplification,followed by a repair reaction in which only two of the four bases arepresent. The other two bases can be added as biotinylated derivativesfor easy detection. Target specific sequences can also be detected usinga cyclic probe reaction (CPR). In CPR, a probe having 3′ and 5′sequences of non-specific DNA and a middle sequence of specific RNA ishybridized to DNA that is present in a sample. Upon hybridization, thereaction is treated with RNase H, and the products of the probeidentified as distinctive products that are released after digestion.The original template is annealed to another cycling probe and thereaction is repeated.

[0324] Qbeta Replicase, described in PCT Application No. PCT/US87/00880,incorporated herein by reference, may also be used as still anotheramplification method in the present invention. In this method, areplicative sequence of RNA that has a region complementary to that of atarget is added to a sample in the presence of an RNA polymerase. Thepolymerase will copy the replicative sequence that can then be detected.

[0325] An isothermal amplification method, in which restrictionendonucleases and ligases are used to achieve the amplification oftarget molecules that contain nucleotide 5′-[alpha-thio]-triphosphatesin one strand of a restriction site may also be useful in theamplification of nucleic acids in the present invention.

[0326] Yet another amplification method is described in PCT ApplicationNo. PCT/US93/07138, which is incorporated herein by reference, may beused in accordance with the present invention. This method ofamplification features treating a target sequence with a firstoligonucleotide (that has a complexing sequence sufficientlycomplementary to a 3′-end portion of the target sequence to hybridizetherewith (this alone is termed a primer), and that has a sequence 5′ tothe complexing sequence that includes a sequence which, indouble-stranded form, acts as a promoter for an RNA polymerase (thisarrangement is termed a promoter-primer), and a second oligonucleotide(which is a primer or promoter-primer that has a complexing sequencesufficiently complementary to the complement of the target sequence tohybridize therewith), under conditions in which anoligonucleotide/target sequence complex may be formed and DNA and RNAsynthesis may occur. In this invention, one or both of the first andsecond oligonucleotides is a mixture of a blocked and an unblockedoligonucleotide sequence (blocked oligonucleotides have a modified 3′end to prevent or reduce the rate and/or extent of primer extension by aDNA polymerase), or a mixture of oligonucleotides with different 3′modifications. Such a mixture significantly enhances the efficiency ofthe specific amplification reaction compared to use of only blocked oronly unblocked oligonucleotides.

[0327] The amplification method synthesizes RNA copies of a targetsequence by use of a mixture of blocked and unblocked promoter-primers,or promoter-primers with different 3′ modifications, consistingessentially of the same nucleic acid sequence in a ratio that providesfor lessened non-specific byproducts. The amplification process occursspontaneously and isothermally under a broad range of conditions.

[0328] Still another amplification methods described in GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

[0329] Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS), including nucleic acidsequence based amplification (NASBA) and 3SR Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference. In NASBA, thenucleic acids can be prepared for amplification by standardphenol/chloroform extraction, heat denaturation of a clinical sample,treatment with lysis buffer and minispin columns for isolation of DNAand RNA or guanidinium chloride extraction of RNA. These amplificationtechniques involve annealing a primer which has target specificsequences. Following polymerization, DNA/RNA hybrids are digested withRNase H while double stranded DNA molecules are heat denatured again. Ineither case the single stranded DNA is made fully double stranded byaddition of second target specific primer, followed by polymerization.The double-stranded DNA molecules are then multiply transcribed by anRNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, theRNA's are reverse transcribed into single stranded DNA, which is thenconverted to double stranded DNA, and then transcribed once again withan RNA polymerase such as T7 or SP6. The resulting products, whethertruncated or complete, indicate target specific sequences.

[0330] Davey et al., EPA No. 329 822 (incorporated herein by referencein its entirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

[0331] Miller et al., PCT Application WO 89/06700 (incorporated hereinby reference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, ie., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR” (Frohman, 1990 incorporated by reference).

[0332] Methods based on ligation of two (or more) oligonucleotides inthe presence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention.

[0333] One of the best known amplification methods is the polymerasechain reaction (referred to as PCR™) which is described in detail inU.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and each incorporatedherein by reference in entirety.

[0334] Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

[0335] A reverse transcriptase PCR amplification procedure may beperformed in order to quantify the amount of mRNA amplified. Methods ofreverse transcribing RNA into cDNA are well known and described inSambrook et al., 1989. Alternative methods for reverse transcriptionutilize thermostable, RNA-dependent DNA polymerases. These methods aredescribed in WO 90/07641, filed Dec. 21, 1990, incorporated herein byreference. Polymerase chain reaction methodologies are well known in theart.

[0336] Another method for amplification is the ligase chain reaction(“LCR”), disclosed in EPA No. 320 308, incorporated herein by referencein its entirety. In LCR, two complementary probe pairs are prepared, andin the presence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

[0337] Following any amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989).

[0338] Alternatively, chromatographic techniques may be employed toeffect separation. There are many kinds of chromatography which may beused in the present invention: adsorption, partition, ion-exchange andmolecular sieve, and many specialized techniques for using themincluding column, paper, thin-layer and gas chromatography.

[0339] Amplification products must be visualized in order to confirmamplification of the marker sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

[0340] In one embodiment, visualization is achieved indirectly,Following separation of amplification products, a labeled, nucleic acidprobe is brought into contact with the amplified marker sequence. Theprobe preferably is conjugated to a chromophore but may be radiolabeled.In another embodiment, the probe is conjugated to a binding partner,such as an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

[0341] In one embodiment, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art and can be found inmany standard books on molecular protocols. See Sambrook et al, 1989.Briefly, amplification products are separated by gel electrophoresis.The gel is then contacted with a membrane, such as nitrocellulose,permitting transfer of the nucleic acid and non-covalent binding.Subsequently, the membrane is incubated with a chromophore-conjugatedprobe that is capable of hybridizing with a target amplificationproduct. Detection is by exposure of the membrane to x-ray film orion-emitting detection devices.

[0342] One example of the foregoing is described in U.S. Pat. No.5,279,72 1, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

[0343] VII. Analysis and Manipulation of Amplification Products

[0344] A. Electrophoresis

[0345] The biochemical and electrophoretic manipulations for successfulDNA sequencing are well characterized, but have not been assembled intoa simple automated processing system. The use of siliconphotolithographic fabrication techniques allows components to becompatible, readily assembled as a single device, and inexpensive tomass produce.

[0346] One type of device contains several components: liquid injectionports, self-pumping channels based upon surface-force gradientphenomena, a thermally isolated amplification chamber, a decision splitpoint, and gel electrophoresis channels. Next to, and underneath, thesecomponents are the system detectors and the controlling circuitry.Within this system a sample is injected, moved to a specific location,and the enzymatic sequencing reactions are performed. A portion of thesequencing product is isolated and sent to a preliminary electrophoresisgel for screening. Using the preliminary information, sequence dataacquisition may be optimized by dividing the remaining product betweenelectrophoresis gels having different resolution characteristics.

[0347] 1. Construction of a Miniature Electrophoresis System.

[0348] Existing electrophoresis technology is able to size fractionatesequencing reactions 800 basepairs and greater using gels 50-100 μmthick and 50 cm long. These results are duplicated using micromachined5-100 μm channels. Short channels of about 1 cm length will be linear,longer channels of about 5 cm will use folded columns.

[0349] The invention will use technology for DNA electrophoresis andconstruct the system using microfabrication techniques. The existingtechnology for DNA sequencing has shown that a polyacrylamide gel 400microns thick and 55 cm long can easily provide single-base resolutionof DNA fragments 100-400 base pairs in length when operated in snapshotmode. The invention may duplicate this separation on a micromachinedsubstrate by using a serpentine channel and an etched glass or siliconsubstrate. However, as has been reported in the literature, separationshould be possible on a much smaller gel.

[0350] The first step in miniaturizing this technology is to analyze theresolution obtained in a typical gel and determine how the dimensionsand operating conditions of a smaller unit would affect the resolutionof the migrating bands. A radiograph of a 55 cm long, 400 μm thickpolyacrylamide sequencing gel that was run for 2 h at 2000 V (58 W)using radioactively tagged DNA was used to estimate μ, Δμ, and D_(eff).While the smaller DNA fragments have moved to the end of the gel, the400 bp fragments are only 10 cm down from the sample wells. To the leftof this run is a “G’ reaction run for 5, 10, 15, 30, 45, and 60 min atthe same voltage. The fragments passing the 10 cm point have beenresolved. Thus, if a detector had been placed at 10 cm and monitoredcontinuously (finishline mode), all bands would have been resolved. Byusing a single detector at the end of the gel and monitoring thatdetector continuously, the invention is able to use a shorter overallgel length. The net voltage that needs to be applied to the gel would beless to give the same electric field strength (i.e., for a 10 cm gel,only 360 V need to be applied to get the same field).

[0351] While this qualitative experiment indicates that the peaks shouldbe resolved in only 10 cm for finishline operation, the invention mayuse the definition of resolution to quantitatively describe theoperation for a variety of operating voltages and channel dimensions.

[0352] For a finishline run, the time of the run is just the length ofthe channel divided by the velocity of the slowest DNA fragment: thetime, therefore, is

t=L ²/(μ_(slow) V)  (9)

[0353] where L is the channel length, μ_(slow) is the mobility of theslowest band, and V is the applied voltage. The resolution between bandsmay be defined as $\begin{matrix}{R = {2\frac{\left( {z_{2} - z_{1}} \right)}{\left( {w_{1} + w_{2}} \right)}}} & (10)\end{matrix}$

[0354] where z_(i) is the location of the center of each band and w_(i)is the width of the band (measured at baseline). The width of the peaksmay be approximated by

w _(i)=(32D _(eff,ave) t)^(½)  (11)

[0355] where D_(eff,ave) is the average dispersion coefficient betweenthe two bands. Knowing that the difference in spatial locations (z₂−z₁)is easily calculated from the electrophoretic mobilities, R may berewritten as

R=¼ΔμV/L(t/2D _(eff,ave) t)  (12)

[0356] Plugging Equation (9) into Equation (12) obtains

R=¼(V/(2D _(eff,ave)))^(½)Δμ_(slow)/μ_(slow) ^(½)  (13)

[0357] where D_(eff),ave is the average dispersion coefficient betweenthe two bands and Δ_(slow) is the difference in mobility between theslowest and next slowest fragments.

[0358] Using a radiograph of a 55 cm long, 400 μm thick polyacrylamidesequencing gel that was run for 2 h at 2000 V (58 W) gel, theseequations may calculate whether the 360 volts in the 10 cm gel would beable to obtain 400 bp resolution. The mobility, μ₄₀₀, is given by(dL)/(Vt)=3.1×10⁻⁵ cm²/Vs. Since the 399 and the 400 bp-length fragmentsare 0.03 cm apart (band 399 and 397 are 0.06 cm apart, therefore 399 and400 are ≈0.06/2 cm apart), then Δμ, is ((Δd)L)/(Vt)=9.2×10⁻⁸ cm²/Vs.D_(deff) is more difficult to obtain. If the plot of the width of abands vs, t^(½), D_(eff,ave) may be found, using equation 11, from theslope of the straight line through the points. This was difficult to dofor the 400 bp band but the plot of this information for the 133 bp longfragment may obtain an estimate of D_(eff,ave)=7×10⁻⁸ cm²/s. This valueis significantly above the molecular diffusion coefficient of DNA infree solution (˜10⁻⁹ cm 2/s) and probably represents both the dispersioncaused by the gel matrix and the low resolution of the x-ray film (Langand Coates, 1968). Note that this calculation assumes that the samplewas applied in an infinitesimally small sample volume; the zerointercept on the plot indicates that this is essentially true. Themicrofluidic devices in the MIDAT system may be used to introduce thesample in a similar fashion as what is done on the large scale.

[0359] The invention may calculate the resolution that should be obtainbased on the above approximate and the 360 V in the 10 cm finishlinegel. Using Δμ=10⁻⁷ cm²/Vs, μ=10⁻⁵ cm²/Vs, D_(eff,ave) 10⁻⁸ cm²/s (thisvalue was decreased to compensate for the band broadening caused by thefilm), and Equation 13, R≈1.0. Although an R value of 1 usually definesa adequate separation, peaks can still be resolved at significantlylower R values. This implies that voltages as low as 200 V (R=0.75) maybe used and still achieve adequate resolution. These voltages poselittle problems in either the glass or silicon devices. Note that,D_(eff) in a variety of different media; D_(eff) should be a functionnot only of the matrix used but also of the distance traveled.

[0360] The length of the channel governs the minimum spatial resolutionthe detector must possess; this spatial distance would be equal to theseparation distance between the two most difficult peaks. Knowing thatΔz=ΔμV/L t and using Equations 9 gets

Δz=LΔμ _(slow)/μ_(slow)  (14)

[0361] Using the parameters obtained above, Δz=0.003 L. Thus if theelectrophoresis channel was 10 cm long, the spatial resolution of thedetector must be 0.03 cm (the distance actually obtained between thelast two peaks).

[0362] The thickness of the gel governs both the quantity of DNA in amigrating band and the heat dissipation of the channel. The quantity ofthe DNA in the band must be matched with the sensitivity of thedetector. Based on the fluorescent probes in current sequencing gels,electrophoresis channels 20-100 μm high should not have a problem withsensitivity of the fluorescent detector.

[0363] The temperature profile in the gel must remain constant to avoidviscosity/mobility gradients that could distort the bands. For symmetricgels, the temperature rise in the center of the gel may be calculated bysolving the heat conduction equation with constant-temperature boundaryconditions and a heat generation term. Published solutions are availableand are of the form (Geankoplis, 1993)

ΔT=(SH ²)/(2k)  (15)

[0364] where ΔT is the difference between the surface temperature andthe center of the gel, S is the heat generation per volume in the gel, His the thickness of the gel, and k is the thermal conductivity of thesolution. Knowing the resistivity of the solution in the gel (ρ), onecan obtain an equation for ΔT in terms of operating variables:$\begin{matrix}{{\Delta \quad T} = \frac{\left( {H\quad {V/L}} \right)^{2}}{8k\quad \rho}} & (16)\end{matrix}$

[0365] Assuming a solution resistivity of 500 Ωcm and thermalconductivity of 0.006 W/cm K, ΔT for a typical sequencing gel (0.4 mmthick, 55 cm long run at 2000 volts (˜50 watts)) is less than 0.1° C.Note that this analysis assumes that the wails of the gels were kept atconstant, equal temperatures; microfabricated heaters and temperaturesensors can easily accomplish this. Equation 8 can be rearranged tosolve for the height of the channel:

H≦1.5L/V  (17)

[0366] where V is in volts. Note that this equation was derived for thespecific test gel that was used and for a temperature difference of 0.1°C.; the equation would need to be derived for other gel/polymer systems.Note also that, for their test gel, the equation correctly calculatesthat H≦0.4 mm.

[0367] There are a variety of other considerations for scale-down of DNAsequencing systems. For instance, the mobilities and dispersioncoefficients may be functions of field intensities or channel wallmaterials. However, the analysis presented here provides the frameworkwith which to design micromachined systems.

[0368] 2. Summary of Channel Specifications

[0369] The channel specification will use three basic criteria: First,the resolution will be measured and designed to be above 0.8:

R=¼(V/(_(2D) _(di eff,ave)))^(½Δμ) _(slow)/μ_(slow)  (13)

[0370] Second, the spatial resolution of the detector will be designedto give adequate spatial resolution:

Δz=LΔμ _(slow)/μ_(slow)  (14)

[0371] Third, the height of the channel will be small enough to preventtemperature gradients:

H≦1.5L/V  (17)

[0372] Thus, to sequence a 400 base-pair fragment by standardtechniques, a 55 cm channel that was 400 μm high is used. The inventionmay be able to run the same sample in a 10 run channel that is 100 μmhigh. Both of these designs may be constructed using siliconmicrofabrication.

[0373] The use of very small diameter capillary systems forelectrophoretic separations has been well established since the early1980's (Jorgenson and Lukacs, 1981; Kuhr, 1990). Capillaryelectrophoresis has an enormous theoretical resolving power and has beencommercially applied to a number of analytical systems (Datta, 1990;Gordon et al., 1988). A variety of liquid capillary formats areavailable, having glass, fused silica, coated, and rectangular columns.Automated injectors and high sensitivity detectors are also beingactively developed. More recently, polyacrylamide gel-filled capillarycolumns have become available for use with DNA fragment separation(Mathes and Huang, 1992; Swerdlow et al., 1992; Drossman et al, 1990).

[0374] Standard liquid capillary electrophoresis has been reported as anintegrated silicon chip technology (Manz et al., 1991; Manz et al.,1992; Lammcrink et al., 1993). The channel dimensions of thechip-integrated liquid capillary electrophoresis system wereapproximately 30 μm wide by 10 μm deep. These columns had very longeffective lengths (several cm), and were constructed as spirals on thesurface of the silicon chip. Very high voltages were used in theseparation, on the order of several kilovolts. As a consequence,breakdown of the silicon substrate was seen as a major obstacle toroutine use. As gel columns of the MIDAT may be micromachined channelsetched on silicon chips, however, much lower voltages are required forgel electrophoresis (1 to 10 volts/cm). Glass capillary gelelectrophoresis has been described with 50 μm to 75 μm inner diametertubing, and effective column lengths of from 50 cm to a few millimeters(Pentoncy et al., 1992; Heller and Tullis, 1992). Theoretical models ofDNA electrophoresis have been useful for estimating the matrix porestructure needed for a particular separation application (Datta, 1990;Gordon et al., 1988).

[0375] 3. Electrophoretic Separation

[0376] Following the amplification reaction, the replicated DNAfragments may be transported to the separation system, again by means ofheating-induced surface tension propulsion. In the MIDAT system, theseparation may be performed using a chip-integrated capillary gelelectrophoresis system. Additionally, the switch to a square channeledcapillary obtained from the micromachining process may benefit theseparation process. Turner (1993) has cited many reports that squarecapillaries are advantageous to circular ones due to their highersurface-volume ratio in providing for more effective heat dissipation.

[0377] The gel is polymerized inside a channel that is made entirelyusing thin films. The unpolymerized acrylamide enters the channel from asessile drop using an injection scheme similar to that describedpreviously. After filling, the acrylamide monomers are polymerized insitu by the addition of catalyst or by photoinduction. Channel walls mayneed to be chemically treated to alter the wetting properties or surfacecharge (Kolb and Cerro, 1991).

[0378] The sample may be directly moved to the anode chamber. Thecomplex mixture of reagents in the isothermal amplification reaction,including unincorporated labeled nucleotide monomers, may necessitate apre-electrophoresis separation step. This step may be accomplished bylow-molecular weight dialysis. Microporous membranes may be fabricatedin silicon (Petersen, 1982), and may provide an on-chip dialysismechanism.

[0379] A low voltage field (1-10 V/cm) is applied to the gel column toinduce electrophoretic motion. This field strength will allowfractionation of DNA fragments of the PCR™ size-range in about 10 min ona 1 cm-long column (Pentoncy et al., 1992; Heller and Tullis, 1992).Switched-field electrophoresis schemes may also be used for more rapidDNA fragment discrimination. In order to reduce the area of theseparation stage, the column may be folded. Such arrangement is morecompact and can increase DNA fragment resolution by several fold.

[0380] B. Detection

[0381] 1. Detector Specifications and Construction

[0382] The detection scheme uses previously tested high-sensitivitysemiconductor diode detectors placed about 0.5-1 μm beneath theelectrophoresis stage. A preferred detection structure has alightly-doped diffusion diode (Kemmer, 1980; Wouters and van Sprakelaar1993) suitable for measurements of both β radiation decay from ³²Plabeling isotopes and visible-light wavelength photons from fluorescentlabels. The detector consists of an n+ diffusion onto a lightly-doped100 p-type float zone silicon substrate with resistivity of 100 Ω-cm.The n+ diffusion is buried under the electrophoresis stage and separatedfrom it by a thin dielectric layer or layers. Fluorescence-baseddetection may be performed using these detectors as photodiodes andadding a thin film optical filter layer(s) placed between the gel anddetector.

[0383] The operation of the diffusion detector is as follows. First areverse bias is applied between the n+ and the substrate, creating adepletion region. Due to the low doping of the substrate, the depletionregion is approximately 8 μm deep for a 10 V bias. When a β particle orphoton traverses the depleted area, electron hole pairs are generated.Carriers generated in the depletion region are readily swept to theelectrodes generating a short current burst. When the mean free path ofthe impacting particle is greater than the depletion region, additionalcarriers diffuse through the substrate until they reach the edge of thedepletion region where they are collected back over time. Therefore thedetector current consists of a sharp peak (corresponding to the chargegenerated in the depletion region) followed by a long tail caused by thediffusing carriers. Diffusion detectors are highly sensitive to smallcharge packets, responsive to the initial position of the ionizationcharge, and easily fabricated in silicon. Approximately 50% of thecharge is collected within 1 ns from the decay.

[0384] Because of the close proximity to the gel, these detectors canpinpoint the position of a radioactive decay or light emission eventwithin 0.5 μm inside the gel. In simple radiation detection mode, eachdetector is capable of sensing a single decay of a β particle from a ³²PDNA label yielding an average of 15,000 electrons per event and a chargeof 2.5 fC. However, this extremely small charge packet demands the useof low-noise and low-parasitic capacitance instrumentation amplifiers.Therefore, the detectors will have on-wafer low-capacitance bufferamplifiers implemented in NMOS technology. Effective charge gains of 5V/fC and noise level of 50 electrons are prepared. In existing CCDcharge amplifiers, smaller charge packets have been sensed on siliconfabricated devices at noise levels of 10-18 electrons (Hynecek, 1992).

[0385] The primary improvements to the existing diode detector involveconversion to a narrow wavelength visible light detector. Since thedetector can function as a fluorescence detector as long as theappropriate filters are used, the invention may include the design andconstruction of optical filter materials directly on the siliconsubstrate and diffusion diode. The electrophoresis channels areseparated from the underlying silicon wafer and electronic components bylayers of silicon oxide and silicon nitride (made by low pressurechemical vapor deposition, LPCVD). The same fabrication method may beused for production of optical filters. First, the spectral absorbancecharacteristics of silicon nitride are well known and vary depending onthe stoichiometric ratios of silicon to nitrogen (Philipp, 1993;Macleod, 1986). The LPCVD method allows control of Si:N ratios, and formost ratios, some range of the visible spectrum is completelytransmitted. In addition, stoichiometric SiO₂ is transparent to visiblelight and much of the UV-range, while pure crystalline silicon (Si) isopaque. Second, the sequential layering of silicon oxide and siliconnitride layers of approximately one-quarter the passed wavelength(0.25×λ) can produce narrow wavelength interference filters. Theinvention uses the known optical properties of silicon-based thin filmmaterials to design and construct interference/absorbance filters,including primarily LPCVD deposition of silicon nitride and siliconoxide over the detector. The final filters may require less than 10 μmof material to achieve complete UV blocking.

[0386] Spatial resolution is an essential requirement in detectors usedfor separations. The spatial resolution determines the accuracy in thelocalization of an emission event within the sieving material. Detectorstructure design may assist in determining the position of an emissionsource. Three distinct phenomena affect the detector resolution. First,in order for the position of the impact to faithfully reflect thelocation of the DNA fragment, the distance between the gel and detectormust be small. In the integrated structure, the detector is in directcontact with the gel channel, hence resolution loss through dispersionis minimal. Second, the detector capture width must be small. Thediffusion diode detectors have a capture width of 2 μm and are easilyformed with lithographic techniques.

[0387] Thirdly, to prevent sensing of emissions from adjacent DNAfragments, the detector response must be insensitive to eventsoriginating outside its capture width. The use of a guard ring aroundthe sensing electrode (Belcarz et al., 1970) eliminates spurioussignals. The ring collects charges generated outside the capture rangeof interest, preventing them from interacting with the central detector.The resulting structure is a low-noise, low-leakage detector. Furtherimprovements on the localization are accomplished using charge divisiontechniques (Knoll, 1989; Alberi and Radeka, 1976; Gerber et al.; 1977;Belau et al., 1983;). The position of the source of the emission eventis calculated from the difference in the two outputs V1 and V2. Therespective charges collected on a set of electrodes are used to estimatethe centroid of the decay through the resistive network. Localization ofthe decay event within 0.5 μm may be possible. Other detector structuresare based on MOSFET structures since these devices are directlycompatible with NMOS process.

[0388] 2. Implementation of Detection Circuits

[0389] The charge collected by the diffusion detector from a β particleor photon yields approximately 10⁴ electrons per event. Semiconductorradiation detectors of this type are typically (Knoll, 1989) connectedto electronic amplifiers. The detector is essentially a diode in reversebias subject to a transient pulse of charge lasting a few nanoseconds.The output of the detector is fed directly to a low noise op-amp(typically a JFET buried channel input device) which integrates thepulse of current and generates a step in the potential at its output.The virtual ground of the op-amp maintains a constant potentialdifference across the detector; therefore its output is independent ofparasitics connected to this node. The parasitic cancellation allows theimplementation of most of the circuits off of the device. Any leakagethrough the diode will induce an offset at the op-amp output. Since thecharge packet is very small, the corresponding integrator capacitorshould also be small. A 250 fF capacitor yields a change of 10 mV at theintegrator output. Hence, it will be desirable to make the resistor ashigh as possible to minimize the noise of the circuit and to retain thecapacitor charge as long as possible (a 10 Ω resistor yields a retentiontime of 2.5 sec). Any drifts in the leakage current, such as response toa temperature change, will lead to large drifts in the output voltage ofthe op-amp and may drive the op-amp outside its linear operation regime.

[0390] For the diffusion diode detector circuit, the invention uses anon-wafer circuit that eliminates most of these parasitic effects. Thecircuit consist of a current source depletion load, an enhancementdischarge transistor, inverters linked to a non-inverting amplificationstage, and a low-pass filter. The circuits are fabricated using a 3 μmNMOS process.

[0391] 3. DNA Detection

[0392] An embodiment of the invention is the DNA sample detector. Twoprimary detection schemes are contemplated. First, fluorescent DNAlabels are commercially available and may be detected using optical p-nphotodiodes constructed below the electrophoresis column. The opticaltransducers are very small with areas on the order of 5 pm. Manydetectors may be constructed in a small area, permitting multipledetector sampling of each electrophoresis column, if desired. The signalof each detector may be multiplexed with an on-chip circuit.

[0393] The fluorescent tags may be excited by an external laser scanningsystem or, more simply, a uniform source of light. Special attention isrequired for wavelength discrimination and detection of the faintfluorescent signals. For ethidium bromide-stained DNA, the excitationwavelength is 302 nm and fluorescent emission is 590 nm. The siliconnitride base of the electrophoresis channel absorbs all wavelengths lessthan −500 nm; thus blocking the UV radiation and transmitting thefluorescent signal. Alternatively, fluorescent DNA labels are becomingavailable with a variety of excitation and emission spectra (Middendorfet al., 1992).

[0394] A method for detection uses radioactively labeled DNA products.Silicon fabricated radiation detectors have been used since the earlier1960's (Bertolini, 1968; Deme, 1971; Knoll, 1979), and are extremelysensitive. The basic structure is similar to that of the p-nphotodetector. The incoming radiation ionizes the silicon creating freecarriers that are collected by the reversed bias diode. The energyneeded to create an electron hole pair is about 3 eV. Typical decayenergies of β-emitting DNA labeling isotopes (³²P, ³³P, ³⁵S) are in the50 to 500 keV region. These energies can create a collected charge of10-13 coulombs per event and an easily detectable current of a fewmicroamps. To prevent the collection of radiation-generated carriersfrom adjacent regions of the chip, a shielding ring is constructedaround the n+detector. The radiation detector may prove less expensivethan the optical scheme, as radioactive DNA tracers are less expensivethan fluorescent labels.

[0395] Amplification may be detected either in the DNA chip or afterremoval of the amplified sample. If the amplified sample is removed forpost-amplification detection (either through the inlet port or through asecond outlet port), the channels and/or chambers are preferably washedwith additional liquid and the wash liquid added to the amplified samplefor detection. If amplification is to be detected within themicrofabricated device, the liquid may be moved through the channels toa separate area containing reagents for a detection reaction and meansfor detecting the amplification products (e.g., labeled probes forhybridization detection and means for detecting the hybridized label ormicroelectrophoresis channels and means for detecting the amplificationproducts by electrophoresis). Alternatively, amplification products maybe detected in the same area where the amplification reaction takesplace when the detection system is compatible with or a component of theamplification reaction, as discussed below. Amplification products maybe detected by hybridization to an assay probe which is typically taggedwith a detectable label. The detectable label may be conjugated to theprobe after chemical synthesis or it may be incorporated into the probeduring chemical synthesis, for example in the form of alabel-derivatized nucleotide. Such labels are known in the art andinclude directly and indirectly detectable labels. Directly detectablelabels produce a signal without further chemical reaction and includesuch labels as fluorochromes, radioisotopes and dyes. Indirectlydetectable labels require further chemical reaction or addition ofreagents to produce the detectable signal. These include, for example,enzymes such as horseradish peroxidase and alkaline phosphatase, ligandssuch as biotin which are detected by binding to label-conjugated avidin,and chemiluminescent molecules. The probes may be hybridized to theirrespective amplification products in solution, on gels, or on solidsupports. Following hybridization, the signals from the associatedlabels are developed, detected and optionally quantitated using methodsappropriate for the selected label and hybridization protocol. Theamount of signal detected for each amplification product may be used toindicate the relative amount of amplification product present. Ligandlabels may also be used on assay probes to facilitate capture of thehybrid on a solid phase (capture probe).

[0396] An alternative method for detecting amplification products is bypolymerase extension of a primer specifically hybridized to the targetsequence. The primer is labeled as described above, for example with aradioisotope, so that the label of the primer is incorporated into theextended reaction product. This method is described by Walker, et al.(1992b) and Walker, et al. (1992a). Another method for detectingamplified target and control sequences is a chemiluminescent method inwhich amplified products are detected using a biotinylated capture probeand an enzyme-conjugated detector probe as described in U.S. Pat. No.5,470,723. After hybridization of these two assay probes to differentsites in the assay region of the target sequence, the complex iscaptured on a streptavidin-coated microtiter plate, and thechemiluminescent signal is developed and read in a luminometer.

[0397] The foregoing detection methods are generally used forpost-amplification detection, either after removing the sample from themicrofabricated device or in a separate detection area of the chipcontaining reagents and detecting means. As another alternative fordetection of amplification products, a signal primer (essentially adetector probe which is extended by polymerase, displaced and rendereddouble-stranded in a target amplification-dependent manner) as describedin EP 0 678 582 may be included in the amplification reaction. In thisembodiment, labeled secondary amplification products are generated in atarget amplification-dependent manner and may be detected as anindication of target amplification in a homogeneous assay format eitherpost-amplification or in real-time (i.e., during amplification). The DNAchip assay formats of the invention are particularly well-suited toreal-time homogeneous amplification detection, as the label of thedetection system (e.g., a signal primer) may be detected through theglass or silica walls of the channel or reaction chamber asamplification is occurring in the liquid aliquot. When the signal primeris labeled with a fluorescent label, the increase in fluorescencepolarization as the signal primer becomes double-stranded may bemonitored in this manner, either in real-time or at a selected endpointin the amplification reaction.

[0398] C. Fluidic and Electronic Integration of the Sequencing System

[0399] Using the invention's micromachined fluid-handling capabilities,they integrate the template preparation, biochemical reactions, andelectrophoresis systems on a single device. Concurrent integration ofelectronic components (detector, heaters, liquid detectors, andtemperature sensors) allows the construction of a self-containedsequencing system. The invention will use sequencing technology in theirmicrofabricated electrophoresis devices. The widths of the channels are(20-100 μm) are on the same order of those currently being producedcommercially and the interior material of the channels is silicon oxide(glass). While most substrates are only 10 cm in diameter (i.e., thelargest linear dimension constructed is 10 cm), longer channels may beconstructed by using a serpentine channel (Jacobson et al., 1994). Bothradiation and fluorescence detectors may be constructed beneath thesechannels to provide either a snapshot (many detectors beneath thechannel or finishline (one detector at the end) mode for runningseparations. The temperature of the gel may also be measured andcontrolled to insure that no gradients exist across the gel. Theseseparation systems may be microfabricated in either silicon or glass.

[0400] 1. Elimination of Sequencing Bottlenecks Using IntelligentSystems

[0401] The system of integrated fluid-handling, electrophoresis,detector, and circuitry components allows feedback and decision-makingdirectly within the device. In one embodiment information-basedprocessing is used to reduce both the systemic and random errors foreach sequencing sample, and to improve reproducibility, error-detection,length of readable data, and compatibility with existing sequencingprotocols.

[0402] The invention assembles individual components for DNA samplehandling and DNA sequencing into increasingly complex, integratedsystems. The incorporation of steps that normally occur in large volumes“on the bench” will reduce the bottlenecks associated with currentlarge-scale DNA sequencing efforts. Since each individual sequencingpreparation, reaction, and electrophoresis run has its own set ofdevices, bottlenecks cannot occur within the integrated system.

[0403] D. Chip Multitasking

[0404] It is contemplated that using micromachining techniques, reactionand separation units that are impossible or impractical to build by anyother techniques are constructed. For instance, an electrophoresischamber with hundreds of DNA detectors along its length may beconstructed for the same cost as constructing a chamber with only onesensor. Also, several hundred of these chambers may be processed on asingle wafer with no additional cost (aside from dicing and otherpost-wafer processing costs). The same technology that makes transistorsin integrated circuits so cheap may allow these complicated, integratedsystems to be produced for a fraction of what their larger-scaleequivalents cost.

[0405] 1. Fluidic Control and Integration

[0406] The controlled movement and mixing of nanoliter drops inmicron-scale channels has been demonstrated using a differential-heatingpropulsion mechanism Control circuitry may maintain uniform biochemicalreaction conditions and to reproducibly measure and detect the locationof individual drops. The individual micro fluidic components for DNAsequencing may maintain compatibility among the devices. A variety ofphotofabricated, integrated DNA analysis systems is contemplated.

[0407] 2. Photolithographic Components as Design Tools

[0408] Once a device component has been developed on a computer aideddesign program, it is replicated across the surface of the wafer as manytimes as desired. Each additional reiteration of the component or groupof components does not cost appreciably more, since the entire wafer isprocessed uniformly. The machines are reproduced photographically. DNAanalysis is a highly repetitive task, requiring many identical devicesgenerating data with very uniform characteristics. Siliconphotolithographic fabrication provides multiple identical devicescheaply.

[0409] 3. Modules for Specific Multi-Step Tasks

[0410] To perform a DNA analysis task, the individual components islinked and function as an integrated device. A set of tasks which areoften found together in a molecular biology protocol are designed as afunctional group or module. The module may be replicated at multiplelocations in the larger device, wherever the specific tasks arerequired. Since each sample has its own set of devices at each step, notime or effort is lost waiting for batch processes to occur, and thereare no points where process bottlenecks occur.

[0411] 4. Incorporation of Earlier Sample Processing Steps in the System

[0412] Increasingly complex devices may be assembled from the individualcomponents and basic functional modules. The modules do not perform anytemplate handling, and consequently require well-characterized templateas starting material. The entire processing stream may be incorporatedonto the device, with all steps included within the silicon-fabricatedenvironment. This embodiment will eliminate process bottlenecks sinceeach sample will have its own dedicated series of instruments.

[0413] As an example of an “intelligent” system, the modules takeadvantage of the ability to hold a sample in reserve, while portion ofthe sample is being examined. The determination of a DNA template sizeand quantity prior to more extensive processing is an use of thiscapability. Size information, for example, can inform the temperature,number of cycles, and electrophoresis conditions of a cycle sequencingrun.

[0414] A single source DNA is used to supply three sequencing reactions.Template DNA is amplified from the source independently for eachsequencing reaction. The template is then divided into two samples andone is assayed for quality and size by gel electrophoresis. Theremaining template is then treated to remove unincorporated primers anddNTPs prior to cycle sequencing. The information obtained by analysis ofhalf of the sample is used to determine the reaction parameters of latersteps. This figure is presented only as an example: alternative templatepreparation strategies are contemplated.

[0415] 5. Reduction of Systemic and Random Error

[0416] Once a fundamental design is established in the microfabricatedformat, it is a minor additional expense to prepare and run additionalgels for each synthesis reaction. Rerun gels are, in fact, one of themajor custom-handling difficulties of current large sequencing groups.As an embodiment, one design would generate two gel reads for eachSanger reaction. Double gel runs, under different conditions, may beable to resolve bands that rnigrate anomalously under a singlecondition. If two parallel gels are run, the output data must then bemerged and compared to resolve the differences between gel reads.

[0417] A second method to reduce error involves duplicate synthesisreaction conditions. For example, longer gel reads are possible (up to1000 bp) using a combination of modified dideoxy:deoxy ratios andextended gel electrophoresis lengths. The original template is dividedin to two samples, one half receiving standard Sanger reaction mix, theother a modified mix which emphasizes longer read length. Both reactionsare then run on sequencing gels, and their output merged. This exampledescribes one possible system developed from individual components: alarge number of alternative strategies are contemplated.

[0418] The MIDAT system is constructed from a conventional silicon waferusing advanced micromechanical fabrication techniques. In certainembodiments, it is contemplated that silicon wafers are constructed with100 to 1000 parallel MIDAT processing units. The multiplex wafer may becapable of simultaneous genotyping an equivalent number of DNA samples,and may provide computer-readable data in less than 3 hours.Miniaturization of DNA analysis results in significant savings inreagents, enzyme, sample handling, and sample processing time.

[0419] As a result of the enormously flexible design characteristics ofsilicon, improved versions may be developed rapidly as the basicbiochemical methods advance. Consequently, other methods of nucleic acidamplification and analysis should be compatible with the MIDAT system.

[0420] 6. Integration of Micromachined Components on a Single Substrate

[0421] It is contemplated the invention will comprise hundreds ofcontrol and detector connections. In practice, the number of externalconnections may be limited by the chip size. By integrating the systemwith on chip electronics, it may be controlled using as little as 5external leads. One embodiment of the invention is on-chip circuitry tocontrol the operation of the MIDAT system. These circuits may beimplemented on the same substrate as the fluidic parts. On-chipintegrated control circuitry may result in a highly compact andefficient design capable of making real-time control decisions. Thesystem may comprise a sample size and flow control circuit, temperaturecycling and timing circuit, electrophoretic separation bias, datadetection and transmission, and a sequencer/timer to control the overalloperation. All the data will be transmitted in serial form between anexternal computer and the MIDAT chip.

[0422] A multicomponent, integrated device includes the elements inFIG. 1. The sections in the diagram represent fundamental processcomponents fabricated on silicon. Sample and reagent are injected intothe device through entry ports or reservoirs (A), and individual liquiddrops are pumped through channels (B) to a thermally controlled reactor,where mixing and restriction enzyme digestion or DNA amplificationoccurs (C). The drop movement is controlled by simple heating, asdifferential heating of the two ends of a drop in a capillary tubeproduces motion (i.e., a thermocapillary pump; Bums et al., 1996). Afterreaction, the biochemical products are moved by the same pumping methodto an electrophoresis channel (D), where DNA migration data arecollected by an integral photodiode (E). The output data are sent offthe integrated device for signal processing and DNA band identification.

[0423] Additional components may be added to the system, provided thechannel connection format remains consistent. Such components maycomprise low temperature polymer-based channels. A silicon wafer withtwo liquid reservoirs, 1000×1000×25 μm), each connected to a 200×25-μmchannel. The channel and reservoir structures are made of alow-temperature polymer (p-xylylene) using a sacrificial etch procedure.Platinum electrophoresis electrodes are visible within each reservoir.Additional platinum surface electrodes and photodiode detectors havebeen placed beneath the channels. The interior channel opening is˜100×25 μm. Peltier cooling surfaces, optical sensors, and ultravioletfilters for continuous spectrophotometric analysis may be present.

[0424] Using photolithographic fabrication, a silicon wafer having >30different electrophoresis channels and integral detectors within a1.25×1.25-cm unit area has been developed. The devices provide areproducible test platform for understanding gel electrophoresis at amicron-size scale (Webster et al., 1996). The silicon components haveprovided considerable preliminary information on channel and detectorformats. The overall arrangement of components across several1.25×1.25-cm DNA processing unit repetitions on a single wafer. Withineach unit are ˜30 different electrophoresis channel and photodiodeconfigurations. The components include an opening to one electrophoresischannel, an associated photodiode, buffer reservoir, external contactpoints for electronic control, and connections for the integralelectrodes and photodiodes. The channels are made using a siliconnitride sacrificial etch process and have an interior cross section of40×5 μm.

[0425] VIII. Kits

[0426] All the essential materials and reagents required for the variousaspects of the present invention may be assembled together in a kit. Thekit generally will comprise reagents to provide the necessary reactionmixture for nucleic acid amplification, including polymerases,nucleotides, buffers, solvents, nucleases, endonucleases, primers,target nucleic acids including DNA and/or RNA, salts, and other suitablechemical or biological components, and a microfabricated substratedefining at least a first channel connected to an isothermally regulatedreaction chamber. One or more of the reagents for the reaction mixturemay be contained in the microfabricated device and/or in a separatereservoir. When the components of the kit are provided in one or moreliquid solutions, the liquid solution is preferably an aqueous solution,with a sterile aqueous solution being preferred.

[0427] In a particularly preferred embodiment, the components of the kitmay also be provided in dried or lyophilized forms. When reagents orcomponents are provided as a dried form, reconstitution generally is bythe addition of a suitable solvent. It is envisioned that the solventalso may be provided in another container means. The kits of theinvention may also include an instruction sheet defining the use of themicrofabricated substrate to amplify nucleic acids.

[0428] The kits of the present invention also will typically include ameans for containing the reagent vials and microfabricated substrate inclose confinement for commercial sale such as, e.g., injection orblow-molded plastic containers into which the desired vials areretained.

[0429] IX. Diagnostics

[0430] The diagnostic system of the present invention generally involvesdetermining either the type or the amount of a wild-type or mutantnucleic acid segment amplified from a biological sample using thechip-based devices of the invention. The biological sample may be from apatient suspected of having a variety of diseases including cancer.Irrespective of the disease, it will be understood that the detection ofa mutant is likely to be diagnostic of a disease, and that the detectionof altered amounts of the target nucleic acid segment is also likely tohave diagnostic implications, particularly where there is a reasonablysignificant difference in amounts between the patient and samples from anormal subject.

[0431] The type or amount of the target nucleic acid present within abiological sample, such as a tissue sample, may be determined oridentified by means of a molecular biological assay, particularly anisothermal nucleic amplification reaction in a microfabricated substratedefining at least a first channel connected to an isothermally regulatedreaction chamber connected to a nucleic analysis component and adetector for the amplified product. Additionally, any of the foregoingmicrofabricated substrate nucleic acid amplification, processing, anddetection systems may be employed as a diagnostic system in the contextof the present invention.

[0432] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered to function well in the practice of theinvention, and thus may be considered to constitute preferred modes forits practice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes may be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

[0433] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); M (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters);ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters);μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); Ci(Curies); MW (molecular weight); OD (optical density); EDTA(ethylenediamine-tetracetic acid); PAGE (polyacrylamide gelelectrophoresis); UV (ultraviolet); V (volts); W (watts); mA(milliamps); bp (base pair); CPM (counts per min).

EXAMPLE 1

[0434] This example is a minimal fully integrated device and wouldinclude the elements identified in FIG. 1. In the chip format, sampleand reagent are injected into the device through entry ports (FIG. 1A)and the solutions pumped through channels (FIG. 1B) to a thermallycontrolled reactor where mixing and isothermal nucleic acidamplification reactions (SDA, Qβ-replicase, etc.), restriction enzymedigestion, ligation, phosphorylation, dephosphorylation, sequencing,other nucleic acid amplification reactions (e.g. PCR ), or otherenzymatic or chemical reaction known to those of skill in the art occurs(FIG. 1C). The biochemical products may then moved by the same or adifferent pumping method to an electrophoresis channel (FIG. 1D), wherenucleic acid migration data are collected by a detector (FIG. 1E) andexported as electronic information. A component of the system is athermocapillary pump capable of connecting diverse individual elements.

[0435] The microfabricated elements in this example are capable orperforming several processing steps in conventional DNA analysis. Theindividual elements have the potential for combination into a completeDNA genotype analysis processing path. Each component was developedusing only silicon or glass photolithographic production methods. As aconsequence, all components retain the ability to be fabricatedconcurrently on the same substrate wafers. The use of common fabricationmethods allows the assembly of increasingly complex, multicomponent,integrated systems from a small, defined set of standardized elements.Fine control of discrete drop location is only dependent on the densityof individual heating elements or other fluid movement devices along thechannel. Detection of the drop location within the channel may beperformed by using capacitors or conductive wires as sensors. Becausethe thermocapillary pump mechanism requires no external force (otherthan application of heat), it should remain scaleable within a widerange of integrated device sizes. Finally, because each droplet is moveduniquely, devices that incorporate branching pathways or parallel sampleanalysis present no inherent obstacle, other than requiring more complexelectronic control circuitry.

[0436] Thermocapillary Pump

[0437] The thermocapillary pump provides movement of discrete drops inmicron-sized channels with no moving parts or valves. A pumping systembased on individual drop movement has three advantages for DNA and/ornucleic acid analysis: samples may be readily divided and mixed, thesample volume may be determined by measuring the drop length, and eachsample is kept separate, reducing the risk of cross-contamination.

[0438] Motion of discrete liquid samples in micron-sized channels may beaccomplished by differentially heating the drop interfaces (FIG. 5). Inchannels, a pressure difference occurs across the liquid-air interface(i.e., capillary pressure). The pressure difference, ΔP_(c), is afunction of the surface tension and, for rectangular channels, is givenby

ΔP_(c) =P _(atm) −P _(liquid)=(2 cos θ)(1/h+1/w),  (18)

[0439] where θ is the contact angle, h is the channel height, w is thechannel width, and σ is the liquid-vapor interfacial tension given by

σ=σ_(o)(1−bT),  (19)

[0440] where σ_(o) and b are positive constants and T is the temperature(Probstein, 1989). Increasing the temperature on one end of the dropdecreases the surface tension and, therefore, increases the internalpressure on that end. The pressure difference between the two endspushes the drop towards the direction of lower pressure at a rate givenby (ignoring edge effects, h<<w)

<ν<=(h/6 μL)[(σ cos θ)_(a)−(σ cos θ)_(r)],  (20)

[0441] where μ is the viscosity, L is the length of the drop, and thesubscripts a and r refer to the advancing and retreating interfaces,respectively. Note that contact angle hysteresis (θ_(a)≠θ_(r)) requiresa threshold pressure difference for positive motion (Tenan et al., 1982,Dussan, 1979).

[0442] A device capable of moving and mixing nanoliter drops usingdifferential heating was constructed by bonding a surface-etched glasswafer to a silicon substrate. A standard aqueous acid wet-etch is usedto produce channels on the 0.5-min glass wafer having two parallel lanesmerging into a single lane of the same cross-sectional dimensions (a Yshape) with dimensions 500 μm wide and 25 μm deep. Metal heaters arepatterned on the silicon substrate having the same Y format and areprotected from liquid by a thin-film barrier. The elements are designedto match the channel layout and are arrayed as two parallel lanes, each500 μm wide, merging into one lane. The individual heaters consist ofpaired aluminum wires winding across a 500×500 μm region. Broad metalareas on either side of the elements are bonding locations forconnection to external circuitry. The heaters are formed by using aninlay process to prevent defects in the barrier layer. A scanningelectron micrograph of a heater wire in cross section showed thedeposited aluminum, silicon oxide, and silicon nitride layers. Theplasma-enhanced chemical vapor deposition process for forming thesilicon oxide and silicon nitride layers results in an undefinedstoichiometry; therefore, the layers are designated SiO_(x) orSi_(x)N_(y). The width of the aluminum element is 5 μm. Thecomplementary heater and channel wafers are aligned and bonded with anadhesive to form the finished device

[0443] Heater Element Wafer Fabrication

[0444] Heater elements were made with a silicon wafer (p-type, 18-22fl-cm, boron≈concentration≈10¹⁵ cm⁻³) as a substrate for growth of SiO₂thermal oxide (1 μm). A photoresist (AZ-5214-E; Hoescht-Celanese) wasapplied to the wafer and spun at 3000 rpm for 30 sec. The resist waspatterned using a mask (M1) and developed. Reactive ion etching(PlasmaTherm, St. Petersburg, Fla.) was performed to 0.35-μm depth intothe SiO₂ layer at the following conditions: CHF₃, 15 standard cubiccentimeters per minute (sccm); CF₄, 15 sccm; 4 mTorr; dc bias voltage of200 V, 100 W, 20 min. The etch depth was measured by profilometer, and0.35-μm metallic aluminum was electron beam deposited. The resist andoverlying metal were lifted off by development using Microposit 1112Aremover in solution (Shipley, Marlboro, Mass.). The barrier layerscovering the aluminum elements consist of sequentially deposited 1 μmSiO_(x), 0.25 μm Si_(x)N_(y), and 1 μm SiO_(x) using plasma-enhancedchemical vapor deposition. Reactive ion etch was used to etch contactholes to the metal layer using a second mask (M2) with conditions: CHF₃,15 sccm; CF₄, 15 sccm; 4 M Torr; and dc bias, voltage of 200 V, 100 W,120 min. Each heating element used as a temperature sensor wascalibrated by measurement of electrical 5 resistance at 22° C. and 65°C. under constant voltage; intermediate temperatures were estimated bylinear interpolation.

[0445] Channel Wafer Fabrication

[0446] Channels were prepared on 500-μm-thick glass wafers (Dow Corning7740) using standard aqueous-based etch procedures. The initial glasssurface was cleaned and received two layers of electron beam-evaporatedmetal (20 nm chromium, followed by 50 nm gold). Photoresist (Microposit1813) was spun at 4000 rpm for 30 sec, patterned-using a mask (GI), anddeveloped. The metal layers were etched in chromium etchant (Cr-14;Cyantek, Newark, Calif.) and gold etchant (Gold Etchant TFA; Transene,Rowley, Mass.) until the pattern was clearly visible on the glasssurface. The accessible glass was then etched in a solution ofhydrofluoric acid and water (1:1). Etch rates were estimated using testwafers, with the final etch giving channel depths of 20-30 μm. For eachwafer, the depth of the finished channel was determined using a surfaceprofilometer. The final stripping steps removed the remainingphotoresist material (PRS-2000; J. T. Baker) and metal layers (Cr-14 andGold Echant TFA).

[0447] Glass-to-Silicon Wafer Bonding and Channel Pretreatment

[0448] Channels etched on glass were bonded to the heater element waferusing a thin film of applied optical adhesive (SK-9 Lens Bond; SumersLaboratories, Fort Washington, PA). The bond was cured under a UV lightsource (365 nm) for 12-24 h. Tests of cured adhesive samples indicatedlittle or no inhibition of restriction endonuclease or thermostable DNApolymerase. Prior to each drop-motion study, the bonded channels wereprepared by washing with ≈100 μl each of the following solutions inseries: 0.1 M NaOH, 0.1 M HCl, 10 mM Tris. HCl (pH 8.0), deionized H₂O,Rain-X Anti-Fog (Unelko, Scottsdale, Ariz., and bovine serum albumin at500 μg/ml (restriction enzyme grade; GIBCO/BRL).

[0449] Movement and Mixing of Liquid Sample

[0450] Two 80-nl drops at their starting locations in the branches ofthe Y-channel; the hydrophilic surface of the channel allows the processto occur spontaneously. The drop volumes are 60 nl and are calculatedfrom the drop length and the known channel cross section. Activating theheaters under the left interfaces propels the drops forward to thechannel intersection where they meet and join to form a single largerdrop. The combined drop is stopped by turning off all heating elementsand may be reversed by heating the right interface. Additionally,circulation patterns generated in the drop during motion aid in mixingthe liquid sample studies using the metal elements as both heaters andtemperature sensors demonstrate that a temperature differential of20-40° C. across the drop is sufficient to provide forward motion inthis particular channel.

[0451] Other sample-handling operations may be performed with thisdevice. For example, drop splitting may be accomplished in two ways.First, a drop may be moved from the single channel, past the Y-channelintersection, and into the two separate channels. While the motion ofthe drop is accomplished by heating the retreating interface, the amountof liquid that enters each of the two channels may be controlled, byselectively heating one of the advancing interfaces. The drop willpreferentially move into the less-heated branch channel. Alternatively,splitting may be performed on a drop held in a single channel bylocalized heating at the drop's center until a bubble of water vaporforms. Continued heating of the expanding water-vapor bubble propels thetwo drop-halves in opposite directions. Although an increased gas-phase,pressure is responsible for this latter motion, properly placed, airvents in the channel may allow the split drops to be moved independentlyusing thermocapillary pumping.

[0452] To confirm compatibility of the propulsion system with DNAsamples and enzymes, an integrated system was tested combining dropmotion, sample mixing, and controlled thermal reaction (FIGS. 1A-C). Asample containing plasmid DNA (supercoiled BluescriptSK; Stratagene) wasloaded into one branch of the Y channel, and a second sample containingTaq I restriction enzyme and digestion buffer was loaded into the other.After sample merging by thermocapillary pumping, the combined drop wasmaintained at 65° C. for 10 min using the integral heaters andtemperature sensors. Capillary gel electrophoresis of the reactionproducts confirmed that DNA digestion on the silicon device was similarto reactions performed in a standard polypropylene vessel. The enzymaticreaction occurred by moving two drops (Taq I restriction enzyme andsupercoiled plasmid) down separate channels using the thermocapillarytechnique, combining the drops, and heating the merged sample to 65° C.in the channel. After the reaction, the sample was expressed from themicrofabricated device and analyzed by conventional capillary getelectrophoresis. The electrophoretic chromatogram shows completedigestion products (elution time, 18-22 min) and minor residualundigested DNA (elution time, 34 min).

[0453] Drop Motion and Restriction Enzyme Digestion

[0454] The bonded channel device was placed on a stereoscope stage(Olympus SZ1145), and the contact pads for the heating elements wereconnected to a regulated power supply. Aqueous samples were applied toeach of the Y-channel branches by gently touching a suspended drop tochannel opening and allowing capillary action to draw the sample intothe device. Measurements of drop length in the channel provided a visualcheck of the loaded volumes. Heating of the drops occurred by passing,≈30 V dc through the element in short pulses and observing the movementof the drops. A small detectable reduction in drop volume fromevaporation was noted in each study, usually <30% of the initial droplength. Drop movement was recorded with a Hamamatsu (Middlesex, N.J.)video camera on videotape, and still images were obtained from thevideotape without modification.

[0455] For the restriction enzyme digestion of DNA, a drop containing0.2 unit of Taq I restriction enzyme in reaction buffer (100 mM NaCl/10mM MgCl₂/10 mM Tris-HCl, pH 8.0), 150 nl total volume was introducedinto one branch of a Y-channel while a drop containing 150 nl of 0.1 μgof supercoiled plasmid per μl (Bluescript SK; Stratagene) was introducedinto the other. Following drop motion, digestion occurred by holding thedrop at a previously calibrated 65° C. for 10 min using ≈4 V de. Thesingle channel portion of the device was uniformly heated by using sevencontiguous heater elements, and the temperature was monitored bymeasuring electrical resistance. The electronic control system consistedof a National Instruments (Austin, Tex.) LabView controller and virtualinstrument software operating on an Apple Macintosh 950.

[0456] PCR™ on Silicon Wafer Surfaces

[0457] PCR™ was performed using standard buffer and primer concentrationconditions for Thermus aquaticus DNA polymerase enzyme (Mullis andFaloona, 1987, Amheim and Erlich, 1992). PCR™ temperature profiles wereas follows: 94° C. for 4 min, preincubation; 94° C. for 1 min, 62° C.for 1 min, 72° C. for 1 min, 35 cycles; 72° C. for 10 min, finalextension. The primer set is specific for a portion of the mouse Tfe3locus and produces a 460-bp-amplified product (primer A,5′-TAAGGTATGCCCCTGGCCAC-3′ (SEQ ID NO:1); primer B,5′-AAGGTCAGCACAGAGTCCTCA-3′) (SEQ ID NO:2 (Roman et al., 1992). For eachexperimental run a complete 75-μl reaction mixture was prepared using100 ng of purified genomic mouse DNA as template and divided into threereactions of 25 μl each. The first reaction was maintained at roomtemperature for 2 h; the second was reacted in a thin-wall polypropylenetube under mineral oil and cycled in a standard thermal cycler; and thethird was placed on the surface of the described heater wafer within asmall polypropylene ring (4 mm diameter, 1.5 mm height) and covered withlight mineral oil. Wafer temperatures were determined by measuringchanges in heater element resistance and were controlled by a NationalInstruments LabView controller and software operating on an AppleMacintosh 950. On completion of the reactions, the three samples wereexamined for efficiency of amplification by agarose gel electrophoresisand ethidium bromide staining.

[0458] Capillary Gel Electrophoresis.

[0459] Following PCR™ amplification or restriction enzyme digestion, DNAgenotyping reactions are typically analyzed by gel electrophoresis. Todemonstrate that standard DNA gel electrophoresis can operate inmicron-sized channels identical to those used for drop motion, studieswere performed using etched glass channels bonded to planar quartz.Channels etched on glass were bonded to a quartz microscope slide usingSK-9 optical adhesive and 24-h UV-illuminated curing. A 10% acrylamideelectrophoresis mix (10% acrylamidel0.3% bis-acrylamide/89 mM Tris.HCl/89 mM sodium borate/10 mM EDTA/0.001%N,N,N′,N″-tetramethylethylenediamine/0.01% ammonium persulfate) wasinjected into the channel and allowed to polymerize. Followingpolymerization, the slide was immersed in a horizontal electrophoresisapparatus containing gel running buffer (89 mM Tris-HCl/89 mM sodiumborate/10 mM EDTA). A 50-μl sample of 100 ng of DNA per μ1 (BluescriptSK plasmid digested with Msp I) containing 0.01% YOYO-1 dye (MolecularProbes) was placed at the negative electrode opening of the channel, andcurrent was applied until a green fluorescing band appeared at thebuffer-to-gel interface (12 V/cm, ≈2 min). The remaining DNA solutionwas rinsed away, replaced by running buffer, and electrophoresis wascontinued by applying current at 12 V/cm for 120 min. The gel wasphotographed under an incandescent light source and viewed using anOlympus stereo microscope and Nikon 35 mm camera with no filters.Separation of the component bands in a range of 100-1000 bp is clearlyvisible <1 mm from the buffer reservoir-to-gel interface. The highresolution of the detector (in this case, a conventional stereomicroscope at x 10 magnification) allowed the use of an unusually shortgel, and resolved several migrating bands.

[0460] Capillary gel electrophoresis of DNA samples was performed usinga Beckman P/ACE instrument with a laser-induced fluorescence detectorand 37 cm length, 100 μm diameter, linear polyacrylamide gel capillaryaccording to manufacturer's recommendation. Samples were stained, theninjected electrokinetically using a water-stacking procedure and run at7400 V dc for 45 min.

[0461] Additional DNA Analysis System Components

[0462] Using microfabrication processes compatible with the constructionof the thermocapillary pump channels, a thermal cycling plat-form, a gelelectrophoresis chamber, and a DNA detector were fabricated and tested.PCR™ thermal cycling was performed on a silicon substrate using heatersand temperature sensors from the same processed wafer as thethermocapillary pump. In this thermal reaction chamber device, a groupof four closely spaced heater elements were tested to ensurecompatibility with the standard PCR™ biochemical reactions. The devicesuccessfully amplified a single-copy sequence from total genomic mouseDNA in small aqueous drops (10-25 μl) placed on the processed siliconsurface and covered with mineral oil to prevent evaporation. However,variations in PCR™ amplification efficiency as large as 4-fold wereobserved between repetitions of the study.

[0463] Diffusion Diode Wafer Fabrication

[0464] Integral DNA sensor elements were fabricated on the surface ofsilicon wafers to electronically detect migrating DNA bands. A sensorcapable of detecting decay events from radioactively labeled DNA may befabricated on the surface of silicon wafers as p-n-type diffusion diode.Radiation detection was chosen for the initial device since such diodeshave a high sensitivity, small aperture dimensions, and well-knownfabrication and response characteristics. Testing of the device with³²P-labeled DNA demonstrates that it readily functions as a sensorcapable of detecting single impacting events. For each diode element,the diffusion regions of the central detector are ≈300 gm long and 4 μmwide and guard ring shield the electrodes. This diode, althoughcurrently configured for high-energy β particle detection, can alsooperate as a fluorescent light detector when combined with a matchedfluorophore, wavelength filter, and excitation source.

[0465] Diode detectors were prepared on 200 Ω-cm, (100), boron-doped,p-type silicon wafer substrates. Diffused layers of phosphorus (5×10¹⁴cm⁻²) and boron (1×10 cm⁻²) were ion-implanted onto the sample inlithographically defined regions (mask D1); thermal silicon oxide wasgrown (0.2 μm at 900° C.) over the wafer; and contact holes were etchedto the diffusion layer with buffered hydrofluoric acid solution. A3.3-μm layer of photoresist (Microposit 1400-37) was patterned to definethe metal pads (mask D2); 50-nm chromium followed by 400-nm gold wasevaporated over the resist; and the metallization lifted off the regionsretaining the resist. In some initial radiation sensitivity tests, alayer of photoresist (Microposit 1813) was applied across the wafer andbaked for 110° C. for 30 min to form an aqueous solution barrier.Additional studies used a double layer of plasma-enhanced chemical vapordeposition silicon oxide and silicon nitride as a barrier, similar tothe layers described for the heater-element wafer. Radioactivephosphorus (³²P) decay events were detected using a sample of labeledDNA in PCR™ buffer placed on the barrier layer. To test sensitivity, thedetector was connected to a charge-sensitive preamplifier (model 550A,EV-Products, Saxonburg, PA), followed by a linear shaping amplifier anda standard oscilloscope, and events were computer recorded.

[0466] The resolving ability of DNA gel electrophoresis systems may beimproved by the proximity and narrow width of silicon-based detectorsplaced immediately beneath the gel channel. Microfabricated diodes maybe placed within 1 micron of the gel matrix and can have an aperture of5 microns or less. Since the gel length required for the resolution oftwo migrating bands is proportional to the resolution of the detector,the incorporation of micron-width electronic detectors may significantlyreduce the total gel length required for DNA analysis withoutsacrificing band-reading accuracy.

[0467] Currently, optical methods using efficient fluorophores candetect atto-molar concentrations (corresponding to ≈10⁵ DNA molecules)migrating in capillary channels of 8×50 μm internal cross section(Woolley and Mathies, 1994). Reactions for synthesizing such DNAquantities can reasonably occur in 10 μl. An integrated system designedfor picoliter volumes may require channel dimensions on the order of 10μm²×100 μm (cross section×length). At this size, thousands of individualdevices would occupy a single 100-mm-diameter wafer.

EXAMPLE 2

[0468] Isothermal Amplification in a Silicon Chip

[0469] The compatibility of the isothermal amplification reagents(available from Becton Dickinson), particularly enzymes, with thesilicon DNA chip assay format was investigated. The components of an SDAreaction for amplification of the IS6110 element of Mycobacteriumtuberculosis, except for the enzymes, were assembled externally to thechip, denatured in a boiling water bath for 2 min and cooled to 52° C.for 2 min. The enzymes were added to bring the total volume to 50 μLcontaining 35 mM K₂HPO₄ pH 7.6, 50 mM NaCl; 10 mM TRIS pH 7.6, 9 mMMgOAc₂, 1.4 mM dCTPα S, 0.2 mM TrP, 0.2 mM dGTP, 0.2 mM dATP, 18.5%(v/v) glycerol, 1 mM DTr, 500 ng human DNA, 500 nM SDA primers (SI andS2), 2.5 mM SDA bumpers (B₁ and B₂), 10⁶ M. tuberculosis genomescontaining the IS6110 target, 160 units BsoBI and 13 units exo⁻ Bstpolymerase. The amplification and bumper primers were as follows, withthe BsoBI recognition sequence shown in bold and the IS6110 targetbinding sequence underlined:5′-CGATTCCGCTCCAGACTTCTCGGGTCTACTGAGATCCCCT-3′ (S1) (SEQ ID NO:3)5′-ACCGCATCGAATGCATCTCTCGGGTAAGGCGTACTCGACC-3′ (S2) (SEQ ID NO:4)5′-CGCTGAACCGGAT-3′ (B1) (SEQ ID NO:5) 5′-TCCACCCGCCAAC-3′ (B2) (SEQ IDNO:6)

[0470] A 4 μL sample of the amplification reaction was immediatelyplaced in a 60 μm deep, 5.1 cm long glass channel etched in 7740 PYREX(Dow Coming) and adhered to a silicon chip, filling the entire channel.The channel was open at both ends. The channel chip was placed on aheater element wafer in contact with about one third of the sample, andthe temperature was held at 52° C. for up to 30 min to allow theamplification reaction to proceed. To remove the sample, about 5 μL ofamplification reaction buffer without the enzymes was placed at one endof the channel and the sample was withdrawn from the other end using asequencing pipette tip. This process was repeated four times to wash thechannel. The total volume recovered was about 20 μL. The amplificationreaction was then stopped by boiling in a water bath and amplificationwas detected in a chemiluminescent assay as described in U.S. Pat. No.5,470,723. The biotinylated capture probe and the alkaline phosphataselabeled detector probe used in the assay are described in Spargo, et al.(1993). As a control, the same SDA reaction was performed in a test tubein the conventional manner. Target amplification efficiency wasequivalent in the conventional SDA reaction and on the DNA chip, withamplification of almost a million-fold. This demonstrated that thephysical changes in the environment on the DNA chip, includingtemperature gradients, inhibitors and surface interactions, did notadversely affect the amplification reaction.

[0471] The ability of the separate components of the amplificationreaction to adequately mix within the channels of the DNA chip was theninvestigated. In one study, 160 nL of enzyme mix was placed in a channelprepared as described above. The target was denatured at 95° C. inamplification buffer (3.84 μL) and cooled to 52° C. prior to loadinginto the channel with the enzyme. The total volume of the reaction mixfilled the entire channel. Amplification was allowed to proceed at 52°C. for 16 min and assayed as before. In a second study, the target inamplification buffer (1.5 μL) was loaded into the channel and moved overthe heating element using air pressure. Using the heater element thetemperature was raised to 80° C. for about 10 sec (temperature spiked toabout 95° C.) to denature the target, then cooled to 52° C. The enzymemix (1.5 μL) was added to the channel with the denatured target to fillthe channel. The amplification reaction was performed (15 min reactiontime) and assayed as before. In an additional study, 1 μL of enzyme mixwas loaded into one end of the channel and 1 μL of target inamplification buffer was loaded into the other end. The portion of thechannel containing the target was heated to 80° C. for about 15 sec todenature the nucleic acids (temperature spiked to about 90° C.) andcooled to 52° C. The two samples were brought into contact by applyingair pressure to the open ends of the channel until the target and enzymealiquots moved into contact with each other. The reaction was held at52° C. on the heater element for 15 min, then removed with washing asdescribed above. Chemiluminescent detection of amplification products inall of these studies revealed efficient amplification of the target,indicating adequate mixing and diffusion of the reactants in all channelconfigurations and protocols tested.

EXAMPLE 3

[0472] This example describes approaches to the problem of forming amoisture barrier over electrical elements of the microscale device.Initial prototypes employed 5000 angstroms of aluminum and covered itwith PECVD SiO_(x). Upon testing, it was determined that the liquidswere penetrating this later and destroying the aluminum heatingelements.

[0473] Without clear evidence what was causing this problem, it washypothesized that the step height of the aluminum was causing cracks inthe passivation layer (the oxide). In order to alleviate the crackingproblem, a layer of Si_(x)N_(y) tried between two layers of SiO_(x),with the thought that the additional thickness would overcome thecracking caused by the step height. It did not.

[0474] As a follow-up approach, a thinner layer (500 angstroms) ofaluminum was tried. This gave {fraction (1/10)}th the step height of theoriginal prototype devices. On top of this aluminum, a triple layer ofSiO_(x), Si_(x)N_(y), and SiO_(x) was employed. Moreover, the processfor making the Si_(x)N_(y) layer was changed to one which would give amore dense layer. This appeared to solve the problem. However, thethinner layer of aluminum created a higher resistance which was notacceptable. It was determined that one needed a way to generate thickerlayers of aluminum for lower resistance, yet keep the surface relativelysmooth (planar). An etch back process was used (now called “the inlayprocess”) to accomplish the task. By etching back into a layer ofSiO_(x) depositing aluminum in the resulting cavity, then stripping theresist mask, a surface was obtained with a step height low enough toprevent cracking of the passivation layers.

[0475] It was also discovered that the metal bonding pads were notadhering well to the initial PECVD SiO_(x) layer. To overcome theproblem, the process was modified by using a wet thermal SiO₂ layer.

EXAMPLE 4

[0476] This example describes approaches to enhancing droplet motion bysurface treatment. In this regard, the principle of using surfacetension to cause droplets to move may be applied to either hydrophilicor hydrophobic surfaces. Glass, for instance, is naturally hydrophilicwith a near zero contact angle with water. Because the oxide coating ofthe present invention is made principally of the same material as glass,it was expected that the devices would also exhibit near zero angles. Itwas discovered, however, that the actual construction materials hadcontact angles far from zero, thus enhancing the effects of contactangle hysteresis (discussed in greater detail in Example 3). Forinstance, water gave a contact angle (static) of ˜42° on polyamide, ˜41°on SiO₂ (major component of most glasses), ˜62° on silicone spray. Toenhance the surface effectiveness, several treatment processes for bothhydrophilic and hydrophobic surfaces were tried, as described below.

[0477] To improve the hydrophilicity of a surface, several cleaningprocedures were tried. It has been reported that surface contaminationand/or roughness can reduce the hydrophilicity of surfaces. Therefore, ahigh concentration chromic acid cleaning, a high concentration sulfuricacid cleaning a baking procedure (to 600° C. for 8 h to burn offcontaminates), and surface coatings were tried. The acid cleaningprocedures were not as effective as the baking procedure; however,neither proved to be compatible with the devices since the concentratedacids would attack the aluminum pads and the high temperature could pealthe aluminum (melting pt. ˜660° C.) or break the adhesive bond betweenthe heater chip and the channel.

[0478] Rain-X antifog (commercially available) as a treatment wasobserved to work. This is a surface treatment which makes surfaceshydrophilic. Although, the resulting surfaces may not be 0°, by usingthis coating the entire surface gets treated giving a uniform surfacefor the droplet. Experimentally, it was found that Rain-X antifogtreatments greatly enhanced droplet motion studies using heat. Anothersuch treatment which was tested but which did not work was a materialcalled SilWet. This material is used in the agriculture industry forenhancing the wetting of plants with agricultural sprays.

[0479] To obtain hydrophobic surfaces, capillaries were coated withRain-X and silane treatments. Neither of these gave angles much greaterthan 90°, therefore, would not work with this mechanism. Thesetreatments would have to have given angles 180° to be useful forhydrophobic studies of motion. Eventually, it was discovered that onecould apply a teflon coating that was sufficiently hydrophobic topossibly warrant future tests.

EXAMPLE 5

[0480] This example describes approaches to droplet motion by heattreatment. As noted previously (above), the contact angle on theadvancing end of a liquid droplet in motion (known as the advancingcontact angle) is greater that the that on the receding end (recedingcontact angle). In the case of a hydrophilic surface - such as water onglass - this tends to create a back pressure countering attempts atforward motion by heating the back side of a droplet. This is best shownby a simple model describing laminar flow through a channel.

[0481] Average Flow Through a Circular Channel:

[0482] <v>=−ΔP*[R²/(8 μL]

[0483] where:

[0484] Δ=value at back−value at front end of droplet

[0485] ΔP=(1/R)*(ΔG)=pressure difference between droplet ends

[0486] ΔG=change in surface tension between the ends of the droplet.

[0487] R=channel radius

[0488] L=droplet length

[0489] μ=viscosity

[0490] Also, for water, ΔG=-constant*ΔT, where temperature increaseslower the surface tension of most liquids (constant=0.16 dyn/cm forwater).

[0491] Therefore:

[0492] <v>=−(ΔG)*(1/R)*[R²/(8 μL)]=[−0.16*ΔT*R/(8 μ)]

[0493] where:

[0494] ΔT=T_(back)−T_(front)

[0495] giving: <v>=[0.16*R/(8 μL)]*(T_(back)−T_(front))

[0496] This expression indicates that any heating on the back end of thedroplet (if the front remains at a lower temperature) will cause theliquid droplet to move. This was not the case experimentally, however.By way of studies using glass capillaries, it was found that there was aminimum temperature difference required to move the droplet. This effectis believed to be the result of contact angle hysteresis (CAH). In CAH,the advancing contact angle is greater than the receding contact angleresulting in a sort of back pressure which must be overcome to achievedroplet movement. CAH occurs when the interface is placed in motion(dynamic angles). To account for this effect, it was included in asteady-state (ID) model for flow. For instance, if the advancing angleis 36° and the receding angle is 29° (with the front of the dropletbeing 25° C.), then the back of the droplet would need to be heated to˜60° C. for a 1 mm long droplet in a 20 μm high channel. This is justone example situation.

[0497] It was discovered experimentally, however, that the channeldimension and fluid parameters (other than surface tension) do notaffect whether or not the droplet will move. They do determine themagnitude of motion (if it occurs). What does determine whether motionwill occur or not is the following inequality:G_(front)/G_(back) > (R_(front)/R_(back)) * (cos   β_(back)/β_(front))where : β = contact  angle.

[0498] The present calculations suggest that a ˜35° C. differencebetween the front and back of a droplet should be sufficient to initiatedroplet motion in a system with advancing angles of 36° and recedingangles of 29° in a 20 μm high channel. Experimental testing of actualdevices however, showed that the front of the droplet heats relativelyquickly thus reducing the temperature difference needed for movementbetween the front and the back of the droplet. This effect required theinvention to use higher voltages to obtain droplet motion. Voltagestypically in the range of ˜30° Volts were found to be required to obtainmotion. Further studies showed that the resulting temperature differencewas ˜40° C. between the front and back of the droplet thus corroboratingthe initial determination of the requirements.

[0499] Discrete droplet motion in a micromachined channel structureusing thermal gradients was demonstrated. The device consists of aseries of aluminum heaters inlaid on a planar silicon dioxide substrateand bonded by glue to a wet-etched glass channel (20 μm depth, 500 μmwidth). Liquid samples were manually loaded into the two channels on theleft using a micropipette. Heating the left interface of each dropletpropels it toward the intersection of the channels. At the intersection,the droplets meet and join to form a single larger droplet. Note that,since the channel cross-section is 20 μm×500 μm, the volume of each ofthese droplets may be calculated from their lengths and is approximately50 nanoliters.

[0500] The heaters along the entire surface of the channel allow it tobe used as a thermal reaction chamber in addition to a droplet-motiondevice. The upper droplet in the figure contains a DNA sample, while thelower contains a restriction digest enzyme (TaqI) and digestion buffer.Following sample merging, the combined droplet was maintained at 65° C.for 30 min using the integral heaters and temperature sensors. Thecompleted enzymatic reaction was confirmed by expressing the dropletfrom the right end of the channel and loading it onto a capillary gelelectrophoresis system with a laser-induced fluorescence detector. Thechromatogram produced by the silicon-device sample was similar tochromatograms generated from DNA digests runs in a standardpolypropylene microreaction vessel.

EXAMPLE 6

[0501] This example describes various approaches for bonding channels tothe substrate which contains circuitry for heating and temperaturesensing of the device of the present invention (see discussion oftwo-part construction, above). First attempts involved Polyamide;regular polyamide was unsatisfactory in that it was found the two pieceswould not stick together.

[0502] Follow-up attempts involved a photo-definable Polyamide. Thisproduced a sticky surface, but would not give a perfect seal along thechannel. It was discovered that the solvents released during the finalbaking process were causing pockets in the polyamide layer. An adhesionlayer was needed which would seal by ‘curing’ and not release solvents.

[0503] Several different epoxies and glues were investigated, as listedin Table 1 below. TABLE 1 Adhesive Form Dries Texture Comments  1. DymaxUV Glue Gel Clear Rubbery Cures on UV exposure.  2. Carter's Rubber GooYellow/Clear Rubbery Dries quickly and Cement stringy when thinned.  3.Borden's Krazy Liquid Clear Hard Thin, dries on first Glue contact.  4.UHU Bond-All Gel/Goo Clear Hard Dries quickly and stringy when thin.  5.Dennison Paste Clear Hard Will not flow on Permanent Glue applying.Stick  6. Elmer's Glue-All Thick White Hard Slow drying. (Borden) Liquid 7. Liquid Nails Thin Paste Wood-like Hard Thick, dries quickly whenthinned.  8. Devcon 5-Minute Gel Yellow/Clear Hard Thick, cures on Epoxyabout 5 min.  9. Scotch Double- Tape Clear Rubbery Tape. Stick Tape 10.Dow Corning Thick Gel Frosty Soft Seals but does not High Vacuum bond.Grease 11. Nujol Mineral Oil Liquid Clear Runny Neither seals (PerkinElmer) (doesn't spread on glass) nor bonds. 12. Household Goop Gel/GooClear Rubbery Contact cement which dries stringy. 13. Permatex WeatherGel/Goo Yellow/Clear Rubbery Dries quickly on Strip Cement stringy whenthinned. 14. Thick Gel Super Gel Clear Hard Does not cure on Gluecontact but does quickly. 15. DAP Weldwood Goo Orange/Clear RubberyContact cement Contact Cement which gets stringy when thinned. 16.Scotch (3M) Thin Goo Yellow/Clear Rubbery Spray. “Gooey” Photo Mount butnot stringy. Spray Adhesive 17. Silicone Resin Liquid Clear SmoothSpray. Dries to (spray) Lacquer thin, clear, and (GC Electronics) sealedcoating.

[0504] A preferred glue was a UV cured glue, although the process ofapplying the UV glue is tedious and requires some practice to avoidputting the glue in places where it does not belong, e.g., in thechannels.

[0505] Hydroxide bonding and screen printing of bonding substances wasalso attempted. Another option was glass tape, but the high temperaturesrequired to melt the tape appeared to be too high for the presentdevices.

EXAMPLE 7

[0506] This example describes a nucleic acid amplification reaction on asilicon-based substrate. The established DNA biochemistry steps for PCR™occur within physiological conditions of ionic strength, temperature,and pH. Thus, the reaction chamber components have design limitations inthat there must be compatibility with the DNA, enzymes and otherreagents in solution.

[0507] To assess biocompatability, components were added to a standardPCR™ reaction. The results indicate PCR™ works well with bond-all glue,goop glue, rubber cement, vacuum grease, silicone spray, reaction vialplastic, stainless steel, wire thermocouple, crushed glass, and glasscapillary, but indicated that crystalline silicon, crushed silicon,rubber gasket, polyamide, UV glue, cured silicone sealer, and liquidnails glue may not be the ideal material for biological compatibility.Given these results, it may be desirable to modify the surface of themicromachined silicon substrate with adsorbed surface agents, covalentlybonded polymers, or a deposited silicon oxide layer.

[0508] To form a biologically compatible heating element, a standardsilicon wafer was coated with a 0.5 μm layer of silicon dioxide. Next, a0.3 μm deep, 500 μm wide channel was etched into the silicon oxide andgold or aluminum was deposited (0.3 μm thick). This inlay processresults in a relatively planar surface and provides a base fordeposition of a water-impermeable layer. The impermeable layer is madeby a sequence of three plasma enhanced vapor depositions: silicon oxide(SiO_(x)), silicon nitride (Si_(x)N_(y)) and silicon oxide (SiO_(x)).Since the materials are deposited from the vapor phase the precisestoichiometries are not known. A thin metal heater design was used forthis device rather than the doped-silicon resistive heaters previouslydemonstrated for micromachined PCR™ reaction chambers, since the narrowmetal inlay allows viewing of the liquid sample through a transparentunderlying substrate, such as glass or quartz. Also, the use of severalindependent heating elements permits a small number to operate as highlyaccurate resistive temperature sensors, while the majority of elementsare functioning as heaters.

[0509] A device fabricated with metal resistive heaters andoxide/nitride/oxide coating was tested for biological compatibility andtemperature control by using PCR™ amplification of a known DNA templatesample. The reaction was carried out on the planar device using twentymicroliters of PCR™ reaction mix covered with mineral oil to preventevaporation. The reaction mixture was cycled through a standard 35-cyclePCR™ temperature cycling regime using the integral temperature sensorslinked to a programmable controller. Since the reaction volume wassignificantly larger than intended for the original heater design, apolypropylene ring was cemented to the heater surface to serve as asample containment chamber. In all test cases, the presence of amplifiedreaction products indicated that the silicon dioxide surface and theheater design did not inhibit the reaction. Parallel amplificationstudies performed on a commercial PCR™ thermocycler gave similarresults. A series of PCR™ compatibility tests indicated that thereaction on the device is very sensitive to controller settings and tothe final surface material in contact with the sample.

[0510] From the above it should be evident that the present inventionmay be adapted for high-volume projects, such as genotyping. Themicrodroplet transport avoids the current inefficiencies in liquidhandling and mixing of reagents. Moreover, the devices are not limitedby the nature of the reactions, including biological reactions.

EXAMPLE 8

[0511] In this example, a test structure is fabricated. The main part isconstructed from a two mask process with five layers of materials on topof the Si substrate. Proceeding from the lowest to the uppermost layer,the SiO, serves as an insulator between the Si substrate and the othermetal layers, which function as solder pads and heating elements. The Tilayer (250A) is for adhesion of Ni. The layers of Ni (1000 A) and Au(1000 A) act as a diffusion barrier for the solder. The Au layer alsoserves as a wettable pad. Finally, the layer of solder is for bondingtwo substrates together. The solder will melt by heating the metallayers. Another substrate that will be bonded has the same constructionexcept for the solder.

[0512] A thermo-pneumatic microvalve is utilized in the test structure.A corrugated diaphragm is chosen for its larger deflection and highersensitivity. The diaphragm (side length=1000 μm, thickness=3 μm, bosssize length=500 μm boss thickness=10 μm) has a deflection of 27 μM at anapplied pressure of 1 atm. This applied pressure is generated by athermo-pneumatic mechanism, which provides a greater actuation force. Apressure of 1 atm is generated in the cavity between the diaphragm andglass by Freon-11 when it is heated 11° C. above room temperature. Tenmasks are expected to fabricate the microvalve.

[0513] A portion of a silicon substrate is a p-type (100)-oriented Siwafer of normal thickness and moderate doping (>1 cm). The preferredwafer thickness, however, is ordinarily a function of the waferdiameter. The upper surface of the silicon wafer containing substrate islapped, polished and cleaned in the normal and accepted manner.Isotropic etching using reactive ion etching (RIE) forms the diaphragmcorrugations with photoresist as the masking material.

[0514] Deep boron diffusion areas form the rims, center bosses, inletand outlet holes of the finished device. The deposition of shallow borondiffusion areas to form a diaphragm. The various metal layers, includingsolder, are then deposited. The deep and shallow boron diffusionprocesses define the shape of the diaphragm and the etch-stop for thedissolved wafer process.

[0515] Following this, the definition of oxide layer to serve asinsulator of the solder of the finished device. Ti adhesion/Ni/Aubarrier and wettable pads are then deposited. The solder mold of Ni andphotoresist is then defined and the first Ni channel is created bysurface-micromachined using photoresist as sacrificial layers. The Nichannel hole is defined using EDP to remove the sacrificial layers, anddefine an channel hole.

[0516] A second Ni channel is defined by Ni and photoresist, and inletand outlet holes are defined using EDP to remove the sacrificial layers.

[0517] Lastly, a Ti/Pt heater in glass is anodically bonded to thesilicon substrate. Freon fills the cavity through a hole in the glasssubstrate. This hole is created from a diamond drill bit and sealed withepoxy.

EXAMPLE 9

[0518] In this example, a low melting point solder was intended to beutilized in the test structure. Because a universally usefulsolder-sealed microvalve will be used in a gas phase microanalyticalsystem, it is not desirable to use a high melting point (m.p.) solder(>200° C.), which might affect the gas properties. In addition, a highm.p. solder may affect other components on the device, such asintegrated circuits, and increase power consumption. As a result, lowmelting point solder is required. Bismuth-bearing solders have thelowest m.p.'s of 47-138° C. However, when a test structure was dippedinto a pool of solder belonging to this group, all the metal layersdissolved into the solution of solder. Moreover, this solder was notselective in wetting the surface of the test structure.

EXAMPLE 10

[0519] In light of the results of the study set forth in Example 7, anattempt was made with commonly available 60:40 Sn:Pb solder (m.p. 183°C.). When the test structure was dipped into a solution of this solder,the metal layers remained intact. Furthermore, these layers demonstratedexcellent wettability for the solder, i e., the solder was confined onlyto the areas of metals.

EXAMPLE 11

[0520] In this example, a device and method for blocking fluid flow in achannel is described. 60:40 Sn:Pb solder, associated with a heatingelement, is placed within a side channel. The heating element at leastpartially liquefies the solder and air flow moves the liquefied solderfrom the side channel into a main channel and cooled, blocking the mainchannel.

EXAMPLE 12

[0521] In this example, a device, which was fabricated using lift-offmethod described above to pattern hydrophobic regions on glass andsilicon substrates, was testing for the separation of water droplets.For the device, a patterned metallic thin film was used to exposeregions that were chosen to be made hydrophobic on a hydrophilicsubstrate. Chromium, Gold or Aluminum was used as the metal layer; thechoice of the metal being based on process compatibility with otherprocessing steps and step height coverage of the etched channels.

[0522] Line widths as narrow as 10 μm were patterned on siliconsubstrates using the methods of the present invention. Water dropsseparated by lines of hydrophobic and hydrophilic regions patterned bythis new technique (the width of the hydrophilic line in the middle is 1mm). The contact angle of water on the OTS (SAM) coated silicon oxidesurface was measured to be approximately 110°.

[0523] One can also define hydrophobic regions in etched channels inglass by performing the lithography using a thick resist. It was foundempirically that cleaning of the substrates prior to immersion in theOTS (SAM) solution is important; improper cleaning results in films thatpartially covers the surface.

EXAMPLE 13

[0524] The results of Example 10, above, demonstrate that hydrophobicand hydrophilic patterns enable one to define and control the placementof aqueous liquids, and more specifically microdroplets of such liquids,on a substrate surface. Use of this patterning technique to split aliquid droplet into multiple liquid droplets. A concentric pattern ofalternating hydrophobic and hydrophilic sectors was imparted to asilicon substrate (the diameter of the circular substrate was 1 cm)using the methods of the present invention as described above. A waterdrop was placed on the pattern and the excess water pulled away using apipet, resulting in multiple drops separated from each other.

EXAMPLE 14

[0525] In this example, studies to position a water front inside achannel using straight channels (depth ranging from 20-40 μm and widthbetween 100-500 μm) with a 500 μm wide hydrophobic region (or patch)patterned a few millimeters away from the side inlet. Water was placedat the inlet using a sequencing pipette (Sigma, least count 0.5 μ1) andwas drawn into the channel by surface forces. The water front stopped atthe hydrophobic patch if a controlled amount of liquid was placed at theinlet. However, if the channels were overloaded, the liquid would tendto overrun the hydrophobic patch. This behavior was prominent in thechannels with smaller cross-section.

[0526] To eliminate the over-running of the liquid over the patches, anoverflow channel was introduced in the design to stop the water runningover the hydrophobic patch (such as that shown FIG. 3). The dimensionsof the channels varied in depth and width as before. Water placed at theinlet is drawn in and splits into two streams at the intersection point.The two fronts move with almost equal velocity until the front in themain channel reaches the hydrophobic patch. The front in the mainchannel stopped at the hydrophobic patch; however, the other frontcontinued to move to accommodate the excess injected water. Using thisoverflow channel design, one can successfully stop aqueous liquids forthe full range of variation in channel dimensions.

EXAMPLE 15

[0527] One embodiment of the device of the present invention (inoperation) utilized a heater. Liquid placed at the inlet stops at thehydrophobic interfaces, and more specifically, stops at theliquid-abutting hydrophobic region. The inlet and overflow ports wereblocked or heavily loaded with excess liquid to ensure that the pressuregenerated acts only in the direction away from the inlet holes. Theheater resistor was actuated by an applied voltage. The flow of currentcaused resistive heating and subsequently increases the air temperaturein the chamber and, therefore, the pressure. After the pressure buildsup to a particular value, a microdrop splits and moves beyond thehydrophobic patch. The drop keeps moving as long as the heater is kepton; the drop velocity decreases as it moves further away. While it isnot intended that the present invention be limited by the mechanism bywhich this takes place, it is believed that the added volume (the volumeby which the drop has moved) brings about a decrease in the pressure.

[0528] To stop or block the moving drop at a location, two strategiesmay be employed. In the first method, the inlet and overflow ports wereopened to the atmosphere and the heater was slowly turned off. Thetemperature inside the chamber falls quickly to around room temperature,thereby reducing the pressure inside the chamber. The water from theinlet flows into the chamber to relieve the pressure. In the secondmethod, a hydrophobic vent was placed away from the chamber to theright. As soon as the moving drop goes past the hydrophobic vent, thedrop stops moving farther. Cooling the chamber to room temperature atthis instant will cause air to flow back through the vent to relieve thelow pressure in the chamber.

[0529] From the above, it should be clear that the compositions, devicesand methods of the present invention permit on-chip actuation usingetched chambers, channels and heaters. There is no requirement formechanical moving parts and the patterns are readily fabricated. Whilethe operations described above have been for simple designs, the presentinvention contemplates more complicated devices involving theintroduction of multiple samples and the movement of multiplemicrodroplets (including simultaneous movement of separate and discretedroplets).

[0530] All of the compositions and/or methods and/or apparatus disclosedand claimed herein may be made and executed without undueexperimentation in light of the present disclosure. While thecompositions and methods of this invention have been described in termsof preferred embodiments, it will be apparent to those of skill in theart that variations may be applied to the compositions and/or methodsand/or apparatus and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

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1 6 20 base pairs nucleic acid single linear 1 TAAGGTATGC CCCTGGCCAC 2021 base pairs nucleic acid single linear 2 AAGGTCAGCA CAGAGTCCTC A 21 40base pairs nucleic acid single linear 3 CGATTCCGCT CCAGACTTCT CGGGTCTACTGAGATCCCCT 40 40 base pairs nucleic acid single linear 4 ACCGCATCGAATGCATCTCT CGGGTAAGGC GTACTCGACC 40 13 base pairs nucleic acid singlelinear 5 CGCTGAACCG GAT 13 13 base pairs nucleic acid single linear 6TCCACCCGCC AAC 13

What is claimed is:
 1. An apparatus for use in the isothermalamplification of a selected nucleic acid, comprising a microfabricatedsubstrate defining at least a first channel, said at least a firstchannel connected to a reaction chamber, and a means for isothermallyregulating the temperature of said reaction chamber.
 2. The apparatus ofclaim 1, wherein said microfabricated substrate further defines at leasta first entry port connected to said at least a first channel.
 3. Theapparatus of claim 1, wherein said microfabricated substrate furtherdefines at least a second channel directly or indirectly connected tosaid reaction chamber.
 4. The apparatus of claim 3, wherein saidmicrofabricated substrate further defines at least a second entry portconnected to said at least a second channel.
 5. The apparatus of claim3, wherein said at least a second channel is connected to said at leasta first channel at a point prior to connection of said at least a firstchannel to said reaction chamber.
 6. The apparatus of claim 1, whereinbiological reagents effective to permit an isothermal nucleic acidamplification reaction are disposed in said reaction chamber, or in afirst or second channel or reservoir that is directly or indirectlyconnected to said reaction chamber.
 7. A method for isothermalamplification of a selected nucleic acid, comprising: a) providing asample comprising said selected nucleic acid, and reagents effective topermit an isothermal amplification reaction, to a microfabricatedsubstrate that defines at least a first channel, said at least a firstchannel connected to an isothermally regulated reaction chamber; and b)conducting an isothermal amplification reaction to amplify said selectednucleic acid.
 8. The method of claim 7, wherein said microfabricatedsubstrate further comprises a flow-directing means system in operablerelation to said at least a first channel.
 9. The method of claim 8,wherein said flow-directing means system is separated from said at leasta first channel by a liquid barrier.
 10. The method of claim 9, whereinsaid liquid barrier comprises a first silicon oxide layer, a siliconnitride layer and a second silicon oxide layer.
 11. The method of claim8, wherein said flow-directing means system comprises a series ofheating elements arrayed along said at least a first channel.
 12. Themethod of claim 11, wherein said heating elements are comprised ofaluminum.
 13. The method of claim 7, wherein said sample is conveyedfrom said at least a first channel to said isothermally regulatedreaction chamber by differential heating of said sample.
 14. The methodof claim 8, wherein said flow-directing means system comprises a seriesof hydrophobic and hydrophilic surface structures arrayed along said atleast a first channel.
 15. The method of claim 14, wherein said at leasta first channel is treated with a hydrophilicity-enhancing compound. 16.The method of claim 14, wherein said at least a first channel ismodified to comprise one or more hydrophobic regions.
 17. The method ofclaim 8, wherein said flow-directing means system comprises a gas sourcein fluid communication with said at least a first channel.
 18. Themethod of claim 7, wherein said microfabricated substrate furtherdefines at least a first entry port connected to said at least a firstchannel.
 19. The method of claim 7, wherein said microfabricatedsubstrate further defines at least a second channel directly orindirectly connected to said reaction chamber.
 20. The method of claim19, wherein said microfabricated substrate further defines at least asecond entry port connected to said at least a second channel.
 21. Thedevice of claim 19, wherein said at least a second channel is connectedto said at least a first channel at a point prior to connection of saidat least a first channel to said reaction chamber.
 22. The method ofclaim 7, wherein said microfabricated substrate further defines at leasta first reservoir directly or indirectly connected to said at least afirst channel or to said reaction chamber.
 23. The method of claim 7,wherein said microfabricated substrate further defines a nucleic acidanalysis component operably connected to said isothermally regulatedreaction chamber.
 24. The method of claim 23, wherein said nucleic acidanalysis component is a gel electrophoresis channel.
 25. The method ofclaim 24, wherein said nucleic acid analysis component is a capillarygel electrophoresis channel.
 26. The method of claim 23, furthercomprising a nucleic acid detection means operably connected to saidnucleic acid analysis component.
 27. The method of claim 26, whereinsaid nucleic acid detection means is a DNA sensor means.
 28. The methodof claim 27, wherein said DNA sensor means detects a radiolabel.
 29. Themethod of claim 28, wherein said DNA sensor means is ap-n-type diffusiondiode.
 30. The method of claim 27, wherein said DNA sensor means detectsa fluorescent label.
 31. The method of claim 30, wherein said DNA sensormeans is a p-n-type diffusion diode combined with a wavelength filterand an excitation source.
 32. The method of claim 7, wherein saidmicrofabricated substrate is comprised of silicon, quartz or glass. 33.The method of claim 7, wherein said reagents comprise reagents forconducting a Strand Displacement Amplification reaction.
 34. The methodof claim 7, wherein said reagents comprise reagents for conducting aself-sustained sequence replication amplification reaction.
 35. Themethod of claim 7, wherein said reagents comprise reagents forconducting a Qβ replicase amplification reaction.
 36. The method ofclaim 7, wherein said reagents are prefabricated into said reactionchamber, or into a first or second channel or reservoir that is directlyor indirectly connected to said reaction chamber.
 37. The method ofclaim 36, wherein said reagents are prefabricated into said reactionchamber, first or second channel or reservoir in a lyophilized form. 38.The method of claim 7, wherein said reagents further comprise a DNAligase.
 39. The method of claim 7, wherein said reagents furthercomprise a nuclease.
 40. The method of claim 39, wherein said reagentsfurther comprise a restriction endonuclease.
 41. The method of claim 7,wherein said sample is derived from an animal having or suspected ofhaving a disease.
 42. The method of claim 41, wherein said sample isderived from a human subject.
 43. A method for detecting the presence ofa selected nucleic acid, comprising: a) introducing a sample suspectedof containing said selected nucleic acid, and reagents effective topermit an isothermal amplification reaction, into a microfabricatedsubstrate defining at least a first channel, said at least a firstchannel connected to an isothermally regulated reaction chamber; b)conducting an isothermally regulated amplification reaction to amplifysaid selected nucleic acid; and c) detecting the presence of theamplified selected nucleic acid, wherein the presence of the amplifiedselected nucleic acid confirms the presence of said selected nucleicacid in said sample.
 44. The method of claim 43, wherein said sample isderived from an animal or human subject.
 45. The method of claim 43,wherein said sample is derived from an animal having or suspected ofhaving a disease, and the presence of said selected nucleic acid isindicative of the disease state.
 46. The method of claim 43, whereinsaid sample is derived from an animal having or suspected of having adisease, and the absence of said selected nucleic acid is indicative ofthe disease state.
 47. The method of claim 43, wherein said selectednucleic acid comprises a mutation not present in the wild-type versionof the selected nucleic acid.
 48. The method of claim 43, wherein saidselected nucleic acid is detected by means of a detectable labelincorporated into the amplified selected nucleic acid by said isothermalamplification reaction.
 49. The method of claim 43, wherein saidselected nucleic acid is detected by means of a labeled nucleic acidprobe.
 50. The method of claim 48, wherein said detectable label is aradioisotopic, enzymatic or fluorescent label.
 51. A method of making adevice for use in isothermal nucleic acid amplification, comprisingpreparing at least a first microfabricated device, chip or waferdefining at least a first channel that is operably connected to anisothermally regulated reaction chamber.
 52. A kit for conductingisothermal amplification of a selected nucleic acid, comprising, insuitable container means: a) at least a first microfabricated substratedefining at least a first channel, said at least a first channelconnected to an isothermally regulated reaction chamber; and b) reagentseffective to permit an isothermal nucleic acid amplification reaction.53. The kit of claim 52, wherein said at least a first microfabricatedsubstrate further defines a nucleic acid analysis component operablyconnected to said isothermally regulated reaction chamber.
 54. The kitof claim 53, wherein said at least a first microfabricated substratefurther comprises a nucleic acid detection means operably connected tosaid nucleic acid analysis component.
 55. The kit of claim 52, whereinsaid reagents effective to permit an isothermal nucleic acidamplification reaction are disposed in said reaction chamber, or in afirst or second channel or reservoir that is directly or indirectlyconnected to said reaction chamber.
 56. A diagnostic system foridentifying a selected nucleic acid, comprising at least a firstmicrofabricated substrate defining at least a first channel that isconnected to at least a first isothermally regulated reaction chamber;said diagnostic system further comprising a nucleic acid analysiscomponent and a nucleic acid detection means in operable associationwith the reaction chamber of said at least a first microfabricatedsubstrate.
 57. The diagnostic system of claim 56, further comprising, inoperable association, at least a second microfabricated substratedefining at least a second channel that is connected to at least asecond isothermally regulated reaction chamber.
 58. The diagnosticsystem of claim 57, wherein said at least a first and at least a secondmicrofabricated substrates are operably connected in series to a singlenucleic acid analysis component and nucleic acid detection means. 59.The diagnostic system of claim 57, wherein said at least a first and atleast a second microfabricated substrates are operably connected inparallel to at least two distinct nucleic acid analysis components andnucleic acid detection means.
 60. The diagnostic system of claim 56,further comprising reagents effective to permit an isothermal nucleicacid amplification reaction.
 61. The diagnostic system of claim 60,wherein said reagents are disposed in said reaction chamber, or in afirst or second channel or reservoir that is directly or indirectlyconnected to said reaction chamber.
 62. A method of making a nucleicacid diagnostic system, comprising preparing at least a firstmicrofabricated substrate defining, in a series of operableassociations, at least a first channel, an isothermally regulatedreaction chamber, a nucleic acid analysis component and a nucleic acidanalysis detection means.